WO2003002737A1 - Novel human topoisomerase 2α inhibitory protein and utilization thereof - Google Patents

Novel human topoisomerase 2α inhibitory protein and utilization thereof Download PDF

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Publication number
WO2003002737A1
WO2003002737A1 PCT/JP2002/006415 JP0206415W WO03002737A1 WO 2003002737 A1 WO2003002737 A1 WO 2003002737A1 JP 0206415 W JP0206415 W JP 0206415W WO 03002737 A1 WO03002737 A1 WO 03002737A1
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amino acid
acid sequence
seq
cells
human
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PCT/JP2002/006415
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French (fr)
Japanese (ja)
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Akira Nakanishi
Fumio Hanaoka
Shinobu Ohmi
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Riken
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a novel human topoisomerase 2 binding factor and use thereof.
  • human topoisomerase 2 ⁇ is referred to as ⁇ 0 ⁇
  • the human topoisomerase 2 ⁇ -binding factor is referred to as ⁇ .
  • the present invention relates to ⁇ capable of interacting with ⁇ to inhibit its activity, an antisense oligonucleotide of the ⁇ , an antibody recognizing the ⁇ la, and a drug using the same.
  • the present invention relates to a method for screening a substance that inhibits the interaction between ⁇ and ⁇ . Background art
  • DNA topoisomerase is present in almost all organisms, from bacteria to humans, and is an essential enzyme for cell growth.
  • type II topoisomerase also referred to as topo II or ⁇ 0 ⁇
  • ⁇ 0 ⁇ is a function that unwinds excessive twisting in DNA due to replication and transcription (relaxation activity), and removes entanglement and entanglement between DNA during chromosome condensation, separation, and distribution (decatenation activity).
  • ⁇ is a basic expression, since its expression kinetics is deeply involved in cell growth and tumorigenesis. He has gained wide interest from the foundation to the clinical field. ⁇ The factors that interact with ⁇ have been energetically investigated, and numerous proteins such as supercoiling factor, p53, casein kinase, c-Jun, sgsl, and Rb protein have been reported. Thus, the discovery of ⁇ function regulators may lead to the discovery of new tumor suppressor gene products related to the cell cycle and the analysis of carcinogenic mechanisms.
  • topoisomerase is a target of anticancer drugs
  • conventional anticancer drugs stop their functions in the form of a clear bubble complex, which is a reaction intermediate with topoisomerase, so that the resulting DNA is cleaved. Occurs. This is considered to be one of the causes of side effects. Finding a regulatory factor of ⁇ 0 ⁇ is useful as a target for new anticancer drugs, and can greatly contribute to the development of anticancer drugs that do not form cleaved complexes and have few side effects.
  • An object of the present invention is to solve the physiological function of ⁇ , which is a protein that binds to ⁇ . Further, the present invention has an object to provide a novel drug based on the physiological function of ⁇ .
  • the present invention utilizes the interaction between Tau0ropai a and Iotataupai alpha, and an object to be achieved by providing a method for screening a new drug.
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and first tried to elucidate the function of ⁇ found from HeLa cells. As a result, ⁇ was found to inhibit ⁇ 0 ⁇ activity, suggesting that ⁇ ⁇ may regulate the function of ⁇ depending on the cell cycle. Was done. Therefore, the present inventors further studied the effects of the physiological function of ⁇ ; particularly the effect of forced expression of ⁇ ; and suppression of the expression by antisense on cells ( ⁇ expression level). The presence or absence of correlation with the expression level of ⁇ ; and the difference in the expression level between normal cells and tumor cells were examined.
  • a human topoisomerase 2 ⁇ inhibitor comprising a human topoisomerase 2 binding factor having any one of the following amino acid sequences: (a) the amino acid sequence of SEQ ID NO: 1; or
  • a human topoisomerase 2 ⁇ inhibitor comprising a protein having a partial amino acid sequence in the amino acid sequence set forth in SEQ ID NO: 1, for example, as set forth in SEQ ID NO: 10.
  • a human topoisomerase 2 ⁇ inhibitor comprising a protein having an amino acid sequence is provided.
  • an apoptosis-inducing agent comprising a human topoisomerase 2 ⁇ -binding factor having any one of the following amino acid sequences:
  • (b) has an amino acid sequence in which one to several amino acids have been deleted, substituted and / or inserted in the amino acid sequence of SEQ ID NO: 1, and interacts with human topoisomerase 2 ⁇ to reduce its activity.
  • a human topoisomerase 2 ⁇ -binding factor having any one of the following amino acid sequences, which is a recombinant protein expressed in insect cells using a baculovirus: Is provided. (a) the amino acid sequence of SEQ ID NO: 1; or
  • amino acid sequence that can be:
  • an antisense oligonucleotide comprising an antisense sequence of any of the following 5 to 100 base sequences in any of the following base sequences.
  • nucleotide sequence in which one to several nucleotides have been deleted, substituted and / or inserted in the nucleotide sequence set forth in SEQ ID NO: 2, and interacting with human topoisomerase 2 ⁇ to inhibit its activity;
  • an inhibitor of human topoisomerase-2 binding factor comprising the antisense oligonucleotide.
  • an expression enhancer for two human topoisomerases comprising the above antisense oligonucleotide.
  • an apoptosis-inducing agent comprising the above antisense oligonucleotide.
  • an anticancer agent comprising the above antisense oligonucleotide.
  • an antibody recognizing a human topoisomerase-2 binding factor having any one of the following amino acid sequences, or a fragment thereof: (a) the amino acid sequence of SEQ ID NO: 1; or
  • a human topoisomerase 2a binding factor having any one of the following amino acid sequences:
  • a screening method for an inhibitor of a binding factor to human topoisomerase 2 comprising measuring an interaction with the enzyme 2 ⁇ and selecting a substance that inhibits the interaction.
  • amino acid sequence of SEQ ID NO: 1 one to several amino acids have a deletion, substitution, Z or insertion amino acid sequence, and interact with human topoisomerase 2 ⁇ to inhibit its activity.
  • Amino acid sequences that can be:
  • the test substance is a small molecule compound, an antibody, an oligonucleotide, or a library thereof.
  • an inhibitor of human Totopoisome hydrolases 2 alpha binding agent is provided.
  • FIG. 1 shows the base sequence and amino acid sequence of ⁇ a.
  • FIG. 2 shows intracellular localization due to overexpression of ⁇ a.
  • FIG. 3 shows detection of apoptosis by overexpression of ⁇ .
  • FIG. 4 shows the construction and purification of a ⁇ a expression system by insect cells.
  • ⁇ antibody column prepared by immobilizing 2.5 rag of purified antibody on CNBr-activated Sepharose 4B (0.5 g)). The eluted fractions (1-8) were dialyzed and subjected to SDS-PAGE and silver staining to confirm protein purification. (B).
  • FIG. 5 shows the inhibitory effect of recombinant ⁇ on ⁇ 0 ⁇ a activity.
  • FIG. 6 shows changes in the expression level of ⁇ in the cell cycle and localization within the cell.
  • Normal human dermal fibroblasts (NB1-RGB) are cultured in a low serum medium (D-MEM containing 0.4% FBS, non-essential amino acids) for 72 hours, and then serum is added to 15% FBS. did.
  • the cells were collected every 4 hours up to 32 hours, taking this time as 0 hour, and the DNA content by laser scanning cytometer was determined by histogram and the G1 / G0, S, G2 / M phase content was calculated as%.
  • A The expression of the ⁇ and T0PI proteins was measured using a simulated knob and their expression levels were measured using NIH Image (B).
  • FIG. 7 shows the examination of the interaction between ⁇ and ⁇ using an anti- ⁇ antibody column.
  • FIG. 8 shows a study of the interaction between ⁇ and ⁇ using plasmon resonance.
  • the measurement by the plasmon resonance method was performed using IAsys plus.
  • ⁇ was immobilized (267 arc seconds) using a CM dextran cuvette as a cuvette for immobilization.
  • T0PII ⁇ was added within the range of 2.9 to 22.2 ⁇ , and this data was analyzed by FASTfit to calculate the association rate constant (kass :), dissociation rate constant (kdiss) and dissociation parallel constant.
  • FIG. 9 shows the expression amount and localization of ⁇ by the antisense oligonucleotide.
  • Sense, sense reverse, random and antisense oligonucleotides were introduced into normal human skin fibroblasts (1 ⁇ 10 6 cells / ml), and 48 hours later, immunoblotting was performed using an anti- ⁇ antibody, and intracellular ⁇ was detected. Was measured. In addition, changes in the expression level of ⁇ were measured by changing the concentration of the antisense oligonucleotide. Furthermore, after introducing the antisense oligonucleotide, the expression levels of ⁇ and ⁇ ⁇ in the cells were measured over time, and changes in the localization of T0PIIa by the antisense oligonucleotide were detected using the indirect tendency antibody method. .
  • Figure 1 0 is, normal cells (human dermal fibroblasts) and cancer cells (Jurcat, HL60, HeLa, renal cancer, rectal cancer, gallbladder cancer, colon cancer) comparisons ⁇ and Tau0roiotaiota alpha expression levels in Show.
  • FIG. 11 shows the effect of the antisense oligonucleotide on normal cells and cancer cells.
  • Liposome 2 / xM antisense against normal cells (NB1-RGB) and cancer cells (HeLa) Introduced by the pectin method. After 48 hours, analysis was performed by LSC (A). At 24, 48, and 69 hours after the introduction of antisense, normal cells and cancer cells were recovered and DNA ladders were detected (B). Furthermore, the presence or absence of PARP cleavage was measured by indirect immunofluorescence on cancer cells 48 hours after transfection with antisense (0).
  • FIG. 12 shows detection of the inhibitory effect of ⁇ ⁇ by the synthetic peptide of ⁇ .
  • the amino acid sequence 132-151 of ⁇ (VTATFPYTTILSIWLATRRV) inhibited ⁇ 0 ⁇ activity in a concentration-dependent manner. Re 1 axation activity was almost completely inhibited at 20 M (A). Decatenation activity was not completely inhibited even at 100 ⁇ M ( ⁇ ), indicating a difference in sensitivity to both activities.
  • the present invention relates to a drug containing ⁇ as an active ingredient, and the drug can be used as a ⁇ 0 ⁇ inhibitor or an apoptosis inducer.
  • ITII c used in the present invention has any of the following amino acids.
  • amino acid sequences (b) one or several amino acids in the amino acid sequence described in SEQ ID NO: 1 have a deleted, substituted and / or inserted amino acid sequence, and may interact with ⁇ 0 ⁇ to inhibit its activity. Possible amino acid sequences:
  • the range of ⁇ 1 to several '' in the ⁇ amino acid sequence in which one to several amino acids have been deleted, substituted and substituted or inserted in the amino acid sequence of SEQ ID NO: 1 '' is not particularly limited.
  • an amino acid sequence capable of interacting with ⁇ 0 ⁇ ⁇ to inhibit its activity refers to a protein having the amino acid sequence described in SEQ ID NO: 1. Means that the protein interacts with ⁇ with the same or higher affinity as a protein having the amino acid sequence of ⁇ to inhibit its activity. Inhibition of ⁇ 0 ⁇ a activity can be assayed by any appropriate method.
  • the inhibition of the activity of ⁇ ⁇ ⁇ ⁇ can be evaluated, for example, by measuring the inhibitory effect of ⁇ on Relaxation activity and Decatenation activity.
  • kinetoplast DNA can be used as the substrate, and for the measurement of the Relaxation activity, the assay can be performed using Supercoiling DNA as the substrate.
  • the inhibitory effect on the activity can be evaluated by, for example, assuming that the amount of DNA after the reaction without adding ⁇ is 100%, and calculating the amount of DNA when adding ⁇ relatively.
  • a protein having a partial amino acid sequence in the amino acid sequence of SEQ ID NO: 1 can be used as a ⁇ inhibitor.
  • the length of the partial amino acid sequence is not particularly limited, and is, for example, from 5 amino acid residues to 100 amino acid residues, preferably from 5 amino acid residues to 50 amino acid residues, and more preferably from 5 amino acid residues to 3 amino acid residues. It is about 0 amino acid residues, particularly preferably about 5 to 20 amino acid residues.
  • a protein having such a partial amino acid sequence for example, a protein having the amino acid sequence of SEQ ID NO: 10 can be mentioned.
  • a protein having such a partial amino acid sequence can be produced by a normal peptide synthesis method using a peptide synthesizer.
  • the method for obtaining the protein having the amino acid sequence of SEQ ID NO: 1 is not particularly limited, and may be a naturally occurring protein, a chemically synthesized protein, or a recombinant protein produced by a genetic recombination technique. Recombinant proteins are preferred because they are relatively easy to operate and can be produced in large quantities.
  • a protein having the amino acid sequence of SEQ ID NO: 1 a protein having the nucleotide sequence encoding the protein (for example, the nucleotide sequence of SEQ ID NO: 2)
  • a target protein can be produced by preparing DNA and introducing it into a suitable expression system.
  • the DNA having the nucleotide sequence of SEQ ID NO: 2 is a suitable primer designed based on the information of the nucleotide sequence of SEQ ID NO: 2 from a human-derived (for example, HeLa cell-derived) cDNA library. It can be obtained by screening using one or a probe. Screening can be performed by plaque hybridization or the like. Alternatively, PCR is performed using a human-derived cDNA library (for example, derived from HeLa cells) as type III and using a suitable primer designed based on the nucleotide sequence information set forth in SEQ ID NO: 2. This allows direct cloning of the target gene.
  • a human-derived cDNA library for example, derived from HeLa cells
  • Expression systems for expressing the recombinant protein are known to those skilled in the art.
  • the DNA is inserted downstream of a promoter in an expression vector, and then the recombinant expression vector is introduced into a host cell suitable for the expression vector.
  • expression vectors for bacteria include pGEMEX-1 (produced by Promega), p QE-9 (produced by QI AG EN), p QE-30 (produced by Q I AG EN), pRSET
  • yeast examples include, for example, YE p13 (ATCC 371 15), YEp 24 (ATCC 37051), Ycp5O (ATCC 374 19), pHS 19, pHS 15 and the like.
  • expression vectors for baculo include pFastBac (manufactured by Gibco BRL), pVL1392 (manufactured by Invitrogen), and the like.
  • PcDNAI pcDM8 (Funakoshi), pcDNAI / AmP (Invitrogen), pREP4 (Invitrogen), and expression vectors for producing recombinant viruses, such as pMFG ( Ta kara And pAd ex (Takara).
  • Promoters which can be used in expression vectors for bacterial e.g., trp promoter (P trp), T7 promoter one, 1 ac promoter (P lac), P L promoter, P R promoter, Ya E. coli, such as P SE promoter
  • Examples include a phage-derived promoter and the like.
  • Examples of promoters that can be used in yeast expression vectors include PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gall promoter, gal10 promoter, heat shock protein promoter, and MF. al promoter, CUP 1 promoter and the like.
  • Examples of a promoter that can be used in an expression vector for baculo include polyhedrin promoter and the like.
  • Examples of a promoter that can be used in an expression vector for animal cells include the promoter of the immediate early (IE) gene of the cytomegalovirus (human CMV), the early promoter of SV40, the promoter of retrowinolas, and the adeno-adenovirus. Winores promoter, metallothionein promoter, heat shock promoter, SRa promoter, actin promoter and the like. Further, the enhancer of IE gene of human CMV may be used together with the promoter.
  • the host cell is not particularly limited as long as it can express the target protein, and includes bacteria, yeast, animal cells, insect cells, and the like. More specifically, bacteria belonging to the genus Etscherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., Kluyveromyces, Saccharomyces, Yeasts belonging to the genus Schizosaccharomyces, Trichosporon, Genus Schiziomyces, Namalba cells, HeLa cells COS 1 cells, COS 7 cells, CHO cells, animal cells such as 293 cells, Sf9, Sf21, And insect cells such as Hive.
  • Methods for introducing a recombinant vector into a host include, for example, a calcium phosphate method, a protoplast method, an electroporation method, and a suffix :! : Roblast method, lithium acetate method, A lipofection method and the like can be mentioned, and it can be appropriately selected according to the type of the host cell.
  • a recombinant protein expressed in insect cells using a baculovirus.
  • the recombinant gene transfer vector and baculovirus are co-transfected into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is transmitted to the insect cells.
  • examples are described in Baculovirus Expression Vectors, A Laboratory Manual; and Current Protocols in 'Molecular' biology, Bio / Technology, 6, 47 (1988), etc.). ).
  • the baculovirus can be obtained, for example, by the use of Autographs californica nuclear polyhedrosis virus, which is a virus that infects Aspergillus insects, such as Autographi californica, nucleus, polyhedrosis virus, and the like.
  • Autographs californica nuclear polyhedrosis virus which is a virus that infects Aspergillus insects, such as Autographi californica, nucleus, polyhedrosis virus, and the like.
  • Examples of a method of co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include a calcium phosphate method and a Lipofexion method.
  • the transformant having the recombinant expression vector having the target DNA prepared as described above is cultured in a medium, the target protein is produced and accumulated in the culture, and the target protein is collected from the culture. Thereby, the recombinant protein can be isolated.
  • the target recombinant protein is expressed in a lysed state in the cells
  • the cells are collected by centrifugation after cell culture, suspended in an aqueous buffer, and then sonicated, a French press, a Mentongaulin homogenizer, and the like.
  • the cells are disrupted using a dynomill or the like to obtain a cell-free extract.
  • a normal protein isolation and purification method that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resins such as getylaminoethyl (DEAE) Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Kasei), and cations using resins such as S-Sepharose FF (manufactured by Pharmacia) Exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose, phenylsepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing, etc.
  • a purified sample can be obtained using a technique such as electrophoresis alone or in combination.
  • a protein having the amino acid sequence of SEQ ID NO: 1 in which one to several amino acids have been deleted, substituted, Z- or inserted has an amino acid sequence represented by SEQ ID NO: 1 and a base sequence represented by SEQ ID NO: 2.
  • Those skilled in the art can appropriately produce the protein based on the sequence information. For example, it can be produced by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques including PCR, mutagenesis, and the like.
  • a mutant DNA can be obtained by using a DNA having the nucleotide sequence of SEQ ID NO: 2 and introducing a mutation into these DNAs.
  • the method can be carried out using a method in which DNA having the base sequence of SEQ ID NO: 2 is brought into contact with a drug as a mutagen, a method of irradiating ultraviolet rays, a genetic engineering technique, or the like.
  • Site-directed mutagenesis one of the genetic engineering techniques, is useful because it is a technique that can introduce a specific mutation at a specific position, and is useful in Molecular Cloning, 2nd edition, Ryrent's Protocols. 'Molecular' biology, Nucleic Acids Research, 10, 6487, 1982, Nucleic Acids Research, 12, 9441, 1984, Nucleic Acids Research, 13, 4431, 1985, Nucleic Acids Research, 13, 8749, 1985, Proc. Natl. Acad. Sci. USA, 79, 6409, 1982, Proc. Natl. Acad. Sci. USA, 82, 488, 1985. Gene, 34, 315, 1985, Gene, 102, 67, 1991, and the like.
  • the target protein can be produced by obtaining DNA having a nucleotide sequence having a mutation in the nucleotide sequence of SEQ ID NO: 2 by the above-described method, and expressing the DNA in the same manner as described above.
  • the agent of the present invention can be used as a ⁇ inhibitor, and its specific use is not particularly limited. For example, it can be used as an apoptosis inducer.
  • Apoptosis was first discovered and defined as morphologically distinct cell death from the classical cell death, necrosis, and subsequent studies have shown that induction and suppression of apoptosis are governed by genes. It has turned out to be so-called programmed cell death. In apoptosis, a complex biochemical reaction occurs along with the activation of cells, producing various protein and DNA degrading enzymes, which act on their own cells to cause cell death. Apoptosis is a physiological cell death that is essential for normal development and differentiation, and occurs in individual cells during normal biological tissue cell rotation. Therefore, it has been shown that excessive reduction of apoptosis causes many dysfunctions.
  • diseases caused by decreased apoptosis include malignant tumors (cancer), leukemias, autoimmune diseases, viral infectious diseases (such as HIV infection), proliferative skin diseases, rheumatoid arthritis, autoimmune diseases, hepatitis, and kidney diseases. And the like. Therefore, the apoptosis-inducing agent of the present invention can be used as a therapeutic or Z- or prophylactic agent for these diseases caused by a decrease in apoptosis.
  • Morphological features of apoptosis include lack of contact with surrounding cells, cytoplasmic enrichment, chromatin condensation and nucleus condensation associated with endonuclease activity, and nuclear segmentation. Disappearance of microvilli on the cell surface and smoothing of the cell surface (bubble formation on the cell surface: membraneblebbing) are also observed. Also, A phenomenon in which DNA is fragmented due to the nuclease activity is also observed, and the cells themselves form cell fragments called apoptotic bodies, and the formed apoptotic bodies are rapidly mobilized by surrounding cells and macrophages. It is phagocytosed and apoptosis occurs. Therefore, apoptosis can be confirmed by, for example, fragmentation of DNA extracted from cells and morphological observation of the cells.
  • the drug of the present invention When used as a medicine, it is generally provided in the form of a pharmaceutical composition containing ⁇ as an active ingredient and a pharmaceutical additive (carrier, excipient, etc.).
  • a pharmaceutical additive carrier, excipient, etc.
  • the agent of the present invention can be administered as a medicament to mammals including humans.
  • the administration route of the drug of the present invention is not particularly limited, and is orally or parenterally administered (for example, intramuscular administration, intravenous administration, subcutaneous administration, intraperitoneal administration, mucosal administration to nasal cavity, or inhalation administration). Any of
  • the form of the drug of the present invention is not particularly limited, and examples of preparations for oral administration include tablets, capsules, fine granules, powders, granules, liquids, syrups, and the like. Preparations include, for example, injections, drops, suppositories, inhalants, transmucosal absorbers, transdermal absorbers, nasal drops, ear drops, and the like.
  • the dose of the drug of the present invention can be appropriately selected in consideration of the gender, age or weight of the patient, the severity of the symptoms, the administration purpose such as prevention or treatment, and the presence or absence of other complications. .
  • the dosage is generally between 0.000 g / kg body weight / day and 0.1000 g / kg body weight / day, preferably between 0.001 / z g / kg body weight / day and 100 g / kg body weight / day.
  • the agent of the present invention is also useful not only as a pharmaceutical, but also as a ⁇ ⁇ ⁇ inhibitor or an apoptosis inducer as an experimental reagent or the like.
  • ⁇ as an active ingredient is generally provided in a form dissolved in an appropriate solvent or the like.
  • the present invention relates to an antisense oligonucleotide comprising an antisense sequence of 5 to 100 consecutive nucleotide sequences in any of the following nucleotide sequences.
  • the range of ⁇ 1 to several '' in the ⁇ base sequence in which one to several bases are deleted, substituted and / or inserted in the base sequence described in SEQ ID NO: 2 '' is not particularly limited, for example, It means about 1 to 60, preferably about 1 to 30, more preferably about 1 to 20, more preferably about 1 to 10, and particularly preferably about 1 to 5.
  • the antisense oligonucleotide of the present invention is a nucleotide that is complementary or hybridizes to a continuous 5 to 100 nucleotide sequence in a DNA sequence encoding any of the above amino acid sequences, It may be a difference between NA and RNA, and may be modified as long as the function is not hindered.
  • antisense oligonucleotide refers to not only those in which all nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are complementary. As long as stable hybridization is possible, there may be some mismatches.
  • Examples of the antisense oligonucleotide of the present invention include an antisense oligonucleotide having a base sequence (TAGCAGGTCCGACAT) shown in SEQ ID NO: 9 in the sequence listing.
  • the antisense oligonucleotide having such a base sequence was able to suppress the expression of ⁇ very effectively.
  • the antisense oligonucleotide used in the present invention suppresses the expression of ⁇ . It is not limited to the above as long as it is possible.
  • the antisense oligonucleotide may be modified. By performing an appropriate modification, the antisense oligonucleotide is less likely to be degraded in vivo, and ⁇ can be more stably inhibited.
  • modified oligonucleotides include S-oligo type (phosphorothioate type), C-5 thiazole type, D-oligo type (phosphodiester type), and ⁇ ⁇ -oligo type (methylphosphonate type).
  • the antisense oligonucleotide may be one in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a zeo atom.
  • Such antisense oligonucleotides are particularly excellent in nuclease resistance, water solubility, and affinity for RNA.
  • Examples of the antisense oligonucleotide in which at least a part of the oxygen atom constituting the phosphate group is substituted and modified with an atom include, for example, S_oligo-type oligonucleotides.
  • the number of bases of the antisense oligonucleotide is preferably 50 or less, more preferably 25 or less. If the number of bases is too large, the labor and cost of synthesis of the oligonucleotide increase, and the yield also decreases. Further, the number of bases of the antisense oligonucleotide is 5 or more, and preferably 9 or more. When the number of bases is 4 or less, the specificity for the target gene is decreased, which is not preferable.
  • the antisense oligonucleotide (or derivative thereof) of the present invention can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available DNA synthesizer (for example, Applied Biostems).
  • the synthesis method can be obtained by a solid phase synthesis method using a phosphoramidite, a solid phase synthesis method using a hydrogen phosphonate, or the like.
  • the antisense oligonucleotide of the present invention comprises an inhibitor of ⁇ , It can be used as a current enhancer, an apoptosis inducer, and an anticancer agent. It is considered that the antisense oligonucleotide of the present invention can exert an anticancer action by specifically inducing apoptosis in cancer cells.
  • the antisense oligonucleotide of the present invention is used as a medicament, it is generally provided in the form of a pharmaceutical composition containing the antisense oligonucleotide and a pharmaceutical additive (carrier, excipient, etc.). You.
  • the antisense oligonucleotide of the present invention can be administered as a medicine to mammals including humans.
  • the administration route of the antisense oligonucleotide of the present invention is not particularly limited.
  • oral administration or parenteral administration for example, intramuscular administration, intravenous administration, subcutaneous administration, intraperitoneal administration, mucosal administration to nasal cavity, or inhalation) Administration, etc.).
  • the formulation of the antisense oligonucleotide is not particularly limited, and examples of formulations for oral administration include tablets, capsules, fine granules, powders, granules, solutions, syrups, and the like.
  • Formulations for administration include, for example, injections, drops, suppositories, inhalants, transmucosal absorbents, transdermal absorbents, nasal drops, ear drops and the like.
  • Those skilled in the art can appropriately select the form of the drug containing the antisense oligonucleotide, the additive for the drug to be used, the method for producing the drug, and the like.
  • an antisense encapsulating material that enhances durability and membrane permeability can be used.
  • ribosome, poly-L-lysine, lipid, cholesterol, ribopectyl or derivatives thereof can be mentioned.
  • the dosage of the antisense oligonucleotide can be appropriately selected in consideration of the patient's sex, age or weight, the severity of the symptoms, the administration purpose such as prevention or treatment, and the presence or absence of other complications. .
  • the dose is generally 0.1 ⁇ g / kg body weight / day to 100 mg Z kg body weight / day, preferably 0.1 ⁇ g Z kg body weight Z day to 1 O mg Z kg body weight / day. It is.
  • Antibodies that recognize ⁇ The antibody of the present invention recognizes ⁇ having any of the following amino acid sequences. (a) the amino acid sequence of SEQ ID NO: 1; or
  • amino acids in the amino acid sequence described in SEQ ID NO: 1 have a deleted, substituted and / or inserted amino acid sequence, and interact with ⁇ to inhibit its activity.
  • Amino acid sequences that can:
  • the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and its production can be performed by a conventional method.
  • a polyclonal antibody that recognizes ⁇ is obtained by immunizing a mammal with ⁇ or a partial peptide thereof as an antigen, collecting blood from the mammal, and separating and purifying the antibody from the collected blood.
  • the antigen may be administered, for example, two to three times at an interval of 7 to 30 S.
  • the dose may be, for example, about 0.05 to 2 mg per antigen.
  • the route of administration is not particularly limited, and can be selected as appropriate from subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc., by injecting intravenously, intraperitoneally or subcutaneously. Preferably, it is administered.
  • the antigen can be used by dissolving it in an appropriate buffer, for example, an appropriate buffer containing a commonly used adjuvant such as complete Freund's adjuvant or aluminum hydroxide, depending on the administration route and conditions. In some cases, no adjuvant is used.
  • the serum of the mammal is sampled, and the antibody titer is measured.
  • booster immunization is performed using, for example, 100 ⁇ g to 100 ⁇ g of the antigen.
  • blood is collected from the immunized mammal, and the blood is collected, for example, by centrifugation, precipitation using ammonium sulfate or polyethylene daricol, gel filtration chromatography, ion exchange. Chromatography such as chromatography and affinity chromatography
  • a polyclonal antibody recognizing the protein of the present invention can be obtained as a polyclonal antiserum by separating and purifying by a conventional method such as graphy.
  • the globulin type of the monoclonal antibody that recognizes ⁇ is not particularly limited, and examples include IgG, IgM, IgA, IgE, and IgD.
  • the cell line that produces the monoclonal antibody is not particularly limited. For example, it can be obtained as a hybridoma by cell fusion between the antibody-producing cell and the Myeoma cell line.
  • the hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.
  • spleen cells As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used.
  • the antigen the protein of the present invention or its partial peptide is used. Mice, rats and the like can be used as immunized animals, and administration of the antigen to these animals is performed by a conventional method. For example, a suspension or emulsion of an adjuvant, such as complete Freund's adjuvant or incomplete Freund's adjuvant, and the protein of the present invention, which is an antigen, is administered several times to the vein, subcutaneous, intradermal, intraperitoneal, etc. of an animal. To immunize animals.
  • an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant
  • spleen cells are obtained as antibody-producing cells from the immunized animal, and the spleen cells are fused with myeloid cells by a known method (G. Kohler et al., Nature, 256 495 (1975)) to obtain a hybridoma. Can be produced.
  • myeloma cell lines used for cell fusion include the P3X63 Ag8, P3U1 and Sp2 / 0 strains in mice.
  • Cell fusion is performed using a fusion promoter such as polyethylene glycol or Sendai virus.Hypoxanthine / aminopterin / thymidine (HAT) medium is used for selection of hybridomas after cell fusion in a conventional manner.
  • Hybridomas obtained by cell fusion are cloned by a limiting dilution method or the like. Further, if necessary, screening by enzyme immunoassay using ⁇ ⁇ ; or a partial peptide thereof can obtain a cell line that produces a monoclonal antibody that specifically recognizes ⁇ . it can.
  • the hybridoma may be cultured by a usual cell culture method or ascites formation method, and the monoclonal antibody may be purified from the culture supernatant or ascites. Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used in an appropriate combination.
  • Examples of the method for immunoassay of ⁇ using the antibody of the present invention include enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, and luminescence immunoassay. .
  • Antibody fragments include F (ab ') 2 fragments, Fab, fragments and the like.
  • labeled antibodies of the above-mentioned antibodies are also within the scope of the present invention. That is, the antibody of the present invention prepared as described above can be labeled and used.
  • the types and methods of labeling antibodies are known to those skilled in the art. For example, enzyme labels such as horseradish peroxidase or alkaline phosphatase, fluorescent labels such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocynate), colloid metal and colored latex labeling with color substances such as roasting two tea labels such Piochin, or 1 2 5 I and isotopic labels, such as can ani gel.
  • enzyme labels such as horseradish peroxidase or alkaline phosphatase
  • fluorescent labels such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocynate)
  • colloid metal and colored latex labeling with color substances
  • Analysis using the labeled antibody of the present invention can be performed by a method well known to those skilled in the art.
  • the present invention is characterized in that in the presence of a test substance, an interaction between ⁇ having any of the following amino acid sequences and ⁇ is measured, and a substance that inhibits the interaction is selected. , ⁇ ⁇ ⁇ ⁇ ⁇ screening method.
  • select a substance that inhibits the interaction for example, select a substance that inhibits the activity of ⁇ ; can do.
  • a specific screening system for example, a screening system for detecting whether or not the activity of ⁇ is restored by adding a substrate D ⁇ , ⁇ , ⁇ and a test substance can be mentioned.
  • test substance used in the present invention any substance can be used, and its type is not particularly limited.
  • specific examples of the test substance may be a low molecular weight compound, an antibody or an oligonucleotide, a natural product extract, or a compound library, a phage display library or a combinatorial library. Construction of compound libraries is known to those skilled in the art, and commercially available compound libraries can also be used.
  • the test substance is preferably a low molecular compound, an antibody, an oligonucleotide, or a library thereof.
  • an inhibitor of ⁇ obtained by the screening method of the present invention is also within the scope of the present invention.
  • the interaction between ⁇ and ⁇ 0 ⁇ is measured in the presence of a test substance.
  • the method for measuring the interaction is not particularly limited. Further, the above-mentioned interaction itself may be directly measured, or the above-mentioned interaction may be indirectly measured by measuring ⁇ activity.
  • ⁇ activity As a method for measuring ⁇ activity, it can be evaluated by, for example, measuring the inhibitory effect of ⁇ on Relaxation activity and Decatenation activity, and the measuring method is as described above.
  • Example 1 In general, the same Atsushi system is performed in the absence of the test substance, and the above-mentioned interaction in the presence and absence of the test substance is measured. It is preferable to determine whether a substance is inhibiting the above interaction.
  • the present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.
  • HeLa, COS- 1, HL60 distributed from the University of Tokyo Medical Research Institute
  • Jurcat distributed from the University of Tokyo Medical Research Institute
  • the following four strains were purchased from RIKEN Genebank and Cell Development Bank: CW-2 (colorectal cancer cell line) ), TUHR4TKB (kidney cancer cell line), TGBC2TKB (gallbladder cancer cell line), TT1TKB (rectal cancer cell line)
  • the HeLa cell-derived cDNA library was converted to type II and PCR was performed using the following primers (30 seconds at 94 ° C, 30 seconds at 94 ° C, 1 minute at 50 ° C, and 2.5 minutes at 72 ° C). The reaction was carried out for 30 minutes, followed by 72 minutes for 3 minutes.
  • Antiserum was collected from rabbits using the amino acid sequence of ITII c 246- GASSRGWDDGDTRSEHSYSESG-267 (peptide 1) (SEQ ID NO: 5) and 302-LWEPTAPEKGKE-313 (peptide 2) (SEQ ID NO: 6) as antigens.
  • a peptide column was prepared using each peptide, and the antibody was purified from the antiserum. From 4 ral antisera, 1.38 mg / ral (5 ml) of the antibody derived from peptide 1 and 2.4 rag / ml (5 ml) of the antibody derived from peptide 2 were prepared.
  • BAC-T0-BAC Baculovirus Expression Systems (Gibco, BRL) was used as the protein expression system.
  • hgyrl gene design the following primers and perform PCR (2 minutes at 94 ° C, 30 cycles at 94 ° C for 30 seconds and 68 ° C for 30 minutes, and a further 3 minutes reaction at 68 ° C) Amplified by Type ⁇ used 10 ng of pRCl.
  • pRC2 Insert the gene of interest into the Rsr II site of the pFASTBAC Htb vector and A fur vector (pRC2) was constructed. 8. 5 ng of pRC2 was introduced into 100 1 DH10BAC competent cells and cultured at 37 ° C for 24 hours. Remove colonies with 2 ml of LB medium
  • SF9 cells were prepared in 1 ⁇ 10 6 cells (6-well plate) in 2 ml of SF-900 II SFM containing 50 units / ml of penicillin and 50 1 streptomycin. After culturing at 27 ° C. for 1 hour, 0, 5, 101 bacmid DNA was dissolved in 100 1 of SF-900 II SF without antibiotics.
  • a peptide antibody was prepared by binding 12 mg of the antibody derived from peptide 2 to CNBr-activated Sepharose 4B (1 ml of swelling). After collecting High Five cells (1 ⁇ 10 8 cells) expressing the target protein, 2 ml of RX buffer (100 raM KC1, 3 mM NaCl, 3.5 mM MgCl 2 , 1.25 mM EGTA, 100 The suspension was suspended in raM HEPES (pH 7.3, 1 mM PMSF) and sonicated.
  • Decatenation activity was measured using kinetoplast DNA (0.175 ⁇ g / assay) as a substrate, and Relaxation activity was measured using pUC19 (0.3 ⁇ g / assay) as a substrate.
  • ⁇ was used in lU / assay (30 ⁇ 1; 50 mM Tris-HCl (pH 7.5), 120 mM KC1, 10 mM MgCl 2 , 0.5 mM ATP, 0.5 mM DTT, 30 ⁇ g / ml nuclease free BSA) The reaction was performed at 37 ° C for 60 minutes.
  • the measurement by the plasmon resonance method was performed using IAsys plus.
  • a CM dextran cuvette was used as a cuvette for solid phase immobilization of ⁇ .
  • Immobilization methods and measuring methods are carried out according to pro tocol IAsys plus accompanying, 267 arc seconds
  • the corresponding immobilized (200 arc seconds 1 ng / mm2) 0 TOPII c of 2 ⁇ 9 to 22 ⁇ 2 nM
  • This data was analyzed within FASTfit, and the association rate constant (kass), dissociation rate constant (kdiss) and dissociation parallel constant were calculated.
  • Apoptosis can be detected by TUNEL method (In situ Apoptosis Detection Kit; Takara Shuzo Co., Ltd.), indirect fluorescent antibody method using an antibody that recognizes PARP cleaved by apoptosis, detection of DNA ladder, Laser Scanning Cytometry (LSC Olympus Optical Co., Ltd.).
  • S-oligo antisense oligonucleotides were designed for residues 1 to 15 of the ⁇ gene.
  • the synthesis was outsourced to BI0GN0STIK.
  • Antisense was introduced into cells using Lipofectin (Gibco, BRL).
  • Antisense was added to 1 ml of serum-free medium to a volume of ⁇ .
  • 25 ⁇ l of Lipofectin was added to another 1 ml of serum-free medium. Both were mixed and allowed to stand at room temperature for 15 minutes. During this time, 1X10 6
  • the cells cultured in the cells 60 culture dishes) were washed twice with PBS (-), and the mixed solution was added dropwise 15 minutes later. The cells were transferred to a 37 ° C 5% CO 2 incubator and cultured for 4 hours. Thereafter, an equal amount of a medium containing serum was added and cultured, and the state of the cells was checked after 24 48 72 hours.
  • NB1-RBG Normal human dermal fibroblasts
  • low serum medium (0.4% FBS, D-MEM containing non-essential amino acids) for 72 hours, and then serum is added to 15% FBS. did.
  • LSC laser scanning cytometer
  • G0 / G1, S, and G2 / M phase contents were determined. 0/0 shown in (FIG. 6 (A)).
  • the expression of ⁇ and ⁇ at each time was detected with anti- ⁇ and anti- ⁇ antibodies, and the protein mass was quantified using NIH Image. (Fig. 6 (B)).
  • ⁇ was localized in the cytoplasm during the G1 / S phase, and cells localized in the nucleus from the G2 / M phase were observed. Therefore, in order to confirm whether ⁇ is present in the nucleus after the G2 / M phase, a nuclear fraction was prepared from the cells 32 hours after the addition of serum, and anti- ⁇ was expressed in the nucleus. The examination was performed using antibodies. As a result, the presence of ⁇ was confirmed in the prepared nuclear fraction (FIG. 6 (D)), suggesting that ITIIct translocated into the nucleus depending on the cell cycle. At this time, the band disappeared by adding an excess amount of the peptide used as the antigen of the anti- ⁇ antibody, and it was revealed that the band detected in the nuclear fraction was equivalent to ⁇ .
  • Antisense oligonucleotide sequencing is generally 15-20 mer, with 20 mer near the ATG codon for translation initiation, 15-20 mer from ATG, or 20 mer containing the boundary between the first exon and the first intron. There are many. However, this time, since the sequence of genomic DNA was not known, we selected the nucleotide sequence of 15-20 mer from ATG. In determining the number of bases, there is a report that a sequence with a GC content of 50% or more in the sequence and a sequence of three or more Gs has a cell growth inhibitory effect other than the antisense effect. Avoided from.
  • the following 15 mer was selected in consideration of the sequence in which the antisense itself does not form a hairpin. 5'-TAGCAGGTCCGACAT-3 '(SEQ ID NO: 9).
  • the prepared antisense was introduced into cells using Lipofectin. Incorporation of the oligonucleotide into cells by Lipofectin was confirmed using a sense oligonucleotide of FITC labeling (antisense reverse complement).
  • ⁇ antisense oligonucleotides were introduced into normal human skin fibroblasts using Lipofectin. 48 hours after transfection, the amount of intracellular ⁇ protein was detected by immunoblot.As a result, ⁇ ⁇ ⁇ ⁇ of antisense-treated cells was reduced by 66% or more compared to the amount of control oligonucleotide-transfected cells. (Fig. 9 ( ⁇ )). In addition, the amount of ⁇ protein decreased in a concentration-dependent manner of the antisense oligonucleotide (FIG. 9 ( ⁇ )).
  • ⁇ was expressed at lower levels in cancer cells than in normal cells, whereas ⁇ ⁇ was expressed at higher levels in cancer cells than in normal cells. Furthermore, it was confirmed that suppressing the expression of ⁇ in normal cells with antisense increases the expression of T0PIIa.
  • HeLa cells and normal human dermal fibroblasts were transfected with antisense oligonucleotides and introduced. The cells 48 hours after the injection were analyzed by LSC, and as a result, GI / Sarrest occurred in the normal cells, but the number of cells did not decrease. In contrast, the number of HeLa cells decreased, and one-third of the cells measured underwent apoptosis (Fig. 11 (A)).
  • ⁇ A synthetic peptide was prepared every 20 residues (10 residues overlap) from the N-terminal side of ⁇ , and the inhibitory effect on ⁇ activity (Relaxation Decatenation activity) was measured.
  • the ⁇ activity was measured in the same manner as in the method described in (viii) Measurement of ⁇ activity in Example 1.
  • the amino acid sequences 132 to 151 (VTATFPYTTILSIWLATRRV) (SEQ ID NO: 10) showed the strongest inhibitory action. The results are shown in FIG. In addition, other than this sequence, a synthetic peptide that inhibits TOPIIc activity was observed.
  • Industrial applicability The present invention, the physiological function of Iotataupai alpha, a protein that binds to TOPIIc is elucidation. According to the present invention, it is possible to provide a novel drug based on the physiological function of ⁇ , and to provide a method for screening a novel drug by utilizing the interaction between ⁇ and ⁇ . It became possible to do.

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Abstract

It is intended to provide novel drugs based on the physiological function of ITIIα. Namely, human topoisomerase 2α inhibitors containing a human topoisomerase 2α-binding factor having any of the following amino acid sequences: (a) the amino acid sequence represented by SEQ ID NO:1; and (b) an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 by deletion, substitution and/or insertion of one to several amino acids and being capable of interacting human topoisomerase 2α to thereby inhibit its activity.

Description

明細書  Specification
新規ヒ トトポイソメラーゼ 2 α阻害蛋白質及びその利用 技術分野  Novel human topoisomerase 2α inhibitory protein and its utilization
本発明は、 新規ヒ トトポイソメラーゼ 2ひ結合因子及びその利用に関する。 以 下、 ヒ トトポイソメラーゼ 2 αを Τ0ΡΠα、 ヒ トトポイソメラーゼ 2 α結合因子 を ΙΤΠαと記載する。 より詳細には、 本発明は、 ΤΟΡΠαと相互作用してその活 性を阻害することができる ΙΤΠα、 該 ΙΤΠαのアンチセンスオリゴヌクレオチ ド、 該 ΠΊ laを認識する抗体、 並びにそれらを用いた薬剤に関する。 さらに本発 明は、 上記 ΙΤΠαと ΤΟΡΠαとの相互作用を阻害する物質をスクリーユングする 方法にも関する。 背景技術  The present invention relates to a novel human topoisomerase 2 binding factor and use thereof. Hereinafter, human topoisomerase 2α is referred to as Τ0ΡΠα, and the human topoisomerase 2α-binding factor is referred to as ΙΤΠα. More specifically, the present invention relates to ΙΤΠα capable of interacting with ΤΟΡΠα to inhibit its activity, an antisense oligonucleotide of the ΙΤΠα, an antibody recognizing the ΠΊla, and a drug using the same. . Further, the present invention relates to a method for screening a substance that inhibits the interaction between ΙΤΠα and ΤΟΡΠα. Background art
DNAトポイソメラーゼは、 細菌からヒ トに至るほとんどすべての生物に存在 し、 細胞の増殖に必要不可欠な酵素である。 特に、 II型のトポイソメラーゼ （ト ポ II又は Τ0ΡΠ とも称する） は、 細胞内で転写、 アポトーシス、 細胞分化、 が ん化などと関連することが明らかにされてきた。 Τ0ΡΠ は、 複製や転写などによ る DNA内の過剰なねじれをほどいたり （relaxation活性）、 染色体の凝縮や分 離、 分配時における DNA間の絡まり、 もつれを除去する （decatenation活性） 機 能を有する。  DNA topoisomerase is present in almost all organisms, from bacteria to humans, and is an essential enzyme for cell growth. In particular, type II topoisomerase (also referred to as topo II or {0}) has been shown to be associated with transcription, apoptosis, cell differentiation, and canceration in cells. {0} is a function that unwinds excessive twisting in DNA due to replication and transcription (relaxation activity), and removes entanglement and entanglement between DNA during chromosome condensation, separation, and distribution (decatenation activity). Have.
大腸菌では、 DNAジャィレース （Τ0ΡΠ) のスーパーコィリング活性 （弛緩 型 DN Aを超らせん型 DN Aに変換させる活性） を阻害するタンパク質 （Gyrl) が見い出されている。 そこで T0PIIの機能を調節するタンパク質がヒ トにも存在 する可能性があると考えて， HeLa細胞から Gyrl相同タンパク質を探索した。 そ の結果、 ΤΟΡΠαと結合し、 その活性を阻害するタンパク質 （ITIIc が見出さ れた。  In Escherichia coli, a protein (Gyrl) that inhibits the supercoiling activity of DNA gyrase ({0}) (the activity to convert a relaxed DNA into a supercoiled DNA) has been found. Therefore, we thought that proteins that regulate the function of T0PII might be present in humans and searched for Gyrl homologous proteins from HeLa cells. As a result, a protein (ITIIc) that binds to ΤΟΡΠα and inhibits its activity was found.
ΤΟΡΠαは、 発現動態が細胞の増殖や腫瘍化に深く関与していることから、 基 礎から臨床的な分野まで広く関心を集めてきた。 ΤΟΡΠ αと相互作用する因子に ついても精力的に調べられ、超らせん化因子、 p53、 casein kinase, c - Jun、 sgsl、 Rb タンパク質など多数のタンパク質が報告されている。 このように、 ΤΟΡΠ αの 機能調節因子を見い出すことは、 細胞周期に関連する新しい癌抑制遺伝子産物の 発見や、 発癌機構の解析につながると考えられる。 ΤΟΡΠα is a basic expression, since its expression kinetics is deeply involved in cell growth and tumorigenesis. He has gained wide interest from the foundation to the clinical field.因子 The factors that interact with α have been energetically investigated, and numerous proteins such as supercoiling factor, p53, casein kinase, c-Jun, sgsl, and Rb protein have been reported. Thus, the discovery of ΤΟΡΠα function regulators may lead to the discovery of new tumor suppressor gene products related to the cell cycle and the analysis of carcinogenic mechanisms.
また、 トポイソメラーゼは、 抗癌剤のターゲットであるが、 従来の抗癌剤はト ポイソメラーゼとの反応中間体であるクリ一バブル複合体の状態でその機能を停 止させるため、 結果的に切断された D N Aを生じる。 これが副作用の要因の一つ と考えられている。 Τ0ΡΠひの調節因子を見い出すことは、 新しい抗癌剤のター ゲットに有用であり、 クリーバブル複合体を形成しない副作用の少ない抗癌剤の 開発に大きく貢献できる。  In addition, topoisomerase is a target of anticancer drugs, but conventional anticancer drugs stop their functions in the form of a clear bubble complex, which is a reaction intermediate with topoisomerase, so that the resulting DNA is cleaved. Occurs. This is considered to be one of the causes of side effects. Finding a regulatory factor of {0} is useful as a target for new anticancer drugs, and can greatly contribute to the development of anticancer drugs that do not form cleaved complexes and have few side effects.
正常細胞の Τ0ΡΠ αの発現量は、 S期で緩やかに増加し、 G2/M期で急激に増加 して M/G1移行期に急速に分解することが報告されている。 一方、癌細胞の Τ0ΡΠ aの発現量は、 細胞周期を通じて常に一定量細胞内に存在することが知られてい る。 正常細胞と腫瘍細胞で TOPI I cの発現量に認められるこのような違いを解明 することは、 ガン化のメカニズムの解析にも発展する可能性を含んでいると考え られる。 発明の開示 It has been reported that the expression level of {0} α in normal cells increases slowly in the S phase, rapidly increases in the G2 / M phase, and rapidly degrades in the M / G1 transition phase. On the other hand, it is known that the expression level of {0} a in cancer cells is always present in a constant amount in cells throughout the cell cycle. Elucidation of such differences in the expression level of TOPI Ic between normal cells and tumor cells may have potential for analysis of the mechanism of canceration. Disclosure of the invention
本発明は、 ΤΟΡΠ αと結合するタンパク質である ΙΤΠ αの生理的機能を解明す ることを解決すべき課題とした。 さらに本発明は、 ΙΤΠ αの生理的機能に基づい た新規な薬剤を提供することを解決すべき課題とした。 さらに本発明は、 Τ0ΡΠ aと ΙΤΠ αとの相互作用を利用して、新規な医薬品をスクリーニングする方法を 提供することを解決すべき課題とした。 An object of the present invention is to solve the physiological function of ΙΤΠα, which is a protein that binds to ΤΟΡΠα. Further, the present invention has an object to provide a novel drug based on the physiological function of ΙΤΠα. The present invention utilizes the interaction between Tau0ropai a and Iotataupai alpha, and an object to be achieved by providing a method for screening a new drug.
本発明者らは上記課題を解決するために鋭意検討し、 先ず、 HeLa細胞から見い 出した ΙΤΠ αの機能の解明を試みた。 その結果、 ΙΤΠ αは Τ0ΡΠ活性を阻害する ことが判明し、 細胞周期に依存して ΤΟΡΠ αの機能を制御している可能性が示唆 された。 そこで、 本発明者らはさらに、 ΙΤΠ α;の生理的機能、 特に ΙΤΠ ο;の強制 発現およびアンチセンスによる発現抑制が細胞 （ΤΟΡΠ αの発現量） に与える影 響、 ΙΤΠ αの発現量と ΤΟΡΠ ο;の発現量との相関性の有無、 さらには正常細胞と 腫瘍細胞におけるその発現量の相違の有無を検討した。 その結果、 ΙΤΠ αの強制 発現により細胞にアポトーシスが誘導されること、 ΙΤΠ aの発現抑制により ΤΟΡΠ αの発現が増大すること、 さらに正常細胞と腫瘍細胞では ΤΟΡΠ αと ΙΤΠ c の発現量が相違していること等を見出した。 本発明はこれらの知見に基づいて 完成したものである。 The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and first tried to elucidate the function of ΙΤΠα found from HeLa cells. As a result, ΙΤΠα was found to inhibit Τ0ΡΠ activity, suggesting that 機能 α may regulate the function of ΤΟΡΠα depending on the cell cycle. Was done. Therefore, the present inventors further studied the effects of the physiological function of ΙΤΠα; particularly the effect of forced expression of ΙΤΠο; and suppression of the expression by antisense on cells (ΤΟΡΠα expression level). The presence or absence of correlation with the expression level of ΤΟΡΠο; and the difference in the expression level between normal cells and tumor cells were examined. As a result, apoptosis is induced in cells by forced expression of ΙΤΠα, 発 現 α expression is increased by suppression of ΙΤΠa expression, and the expression levels of ΤΟΡΠα and ΙΤΠc differ between normal cells and tumor cells. And so on. The present invention has been completed based on these findings.
即ち、 本発明によれば、 下記の何れかのアミノ酸配列を有するヒ トトポイソメ ラーゼ 2 ひ結合因子を含むヒ トトポイソメラーゼ 2 α阻害剤が提供される。 ( a ) 配列番号 1に記載のァミノ酸配列；又は  That is, according to the present invention, there is provided a human topoisomerase 2α inhibitor comprising a human topoisomerase 2 binding factor having any one of the following amino acid sequences: (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のアミノ酸配列において 1から数個のアミノ酸が欠失、 置換及び/又は挿入したアミノ酸配列を有し、 ヒ トトポイソメラーゼ 2 αと相互 作用してその活性を阻害することができるァミノ酸配列：  (b) having an amino acid sequence in which one to several amino acids are deleted, substituted, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and interacts with human topoisomerase 2α to inhibit its activity. Amino acid sequences that can:
本発明の別の態様によれば、 配列番号 1に記載のアミノ酸配列における部分ァ ミノ酸配列を有するタンパク質を含む、 ヒ トトポイソメラーゼ 2 α阻害剤が提供 され、 例えば、 配列番号 1 0に記載のアミノ酸配列を有するタンパク質を含む、 ヒ トトポイソメラーゼ 2 α阻害剤が提供される。  According to another aspect of the present invention, there is provided a human topoisomerase 2α inhibitor comprising a protein having a partial amino acid sequence in the amino acid sequence set forth in SEQ ID NO: 1, for example, as set forth in SEQ ID NO: 10. A human topoisomerase 2α inhibitor comprising a protein having an amino acid sequence is provided.
本発明の別の態様によれば、 下記の何れかのアミノ酸配列を有するヒ トトポィ ソメラーゼ 2 α結合因子を含むアポトーシス誘導剤が提供される。  According to another aspect of the present invention, there is provided an apoptosis-inducing agent comprising a human topoisomerase 2α-binding factor having any one of the following amino acid sequences:
( a ) 配列番号 1に記載のァミノ酸配列；又は  (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のァミノ酸配列において 1から数個のァミノ酸が欠失、 置換及び/又は挿入したアミノ酸配列を有し、 ヒ トトポイソメラ一ゼ 2 αと相互 作用してその活性を阻害することができるァミノ酸配列：  (b) has an amino acid sequence in which one to several amino acids have been deleted, substituted and / or inserted in the amino acid sequence of SEQ ID NO: 1, and interacts with human topoisomerase 2α to reduce its activity. Amino acid sequences that can be inhibited:
本発明のさらに別の態様によれば、 バキュロウィルスを用いて昆虫細胞で発現 させた組み換えタンパク質であることを特徴とする、 下記の何れかのアミノ酸配 列を有するヒ トトポイソメラ一ゼ 2 α結合因子が提供される。 ( a ) 配列番号 1に記載のァミノ酸配列；又は According to still another aspect of the present invention, a human topoisomerase 2α-binding factor having any one of the following amino acid sequences, which is a recombinant protein expressed in insect cells using a baculovirus: Is provided. (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のアミノ酸配列において 1から数個のァミノ酸が欠失、 置換及び Z又は挿入したアミノ酸配列を有し、 ヒ トトポイソメラーゼ 2 αと相互 作用してその活性を阻害することができるアミノ酸配列：  (b) one or several amino acids in the amino acid sequence of SEQ ID NO: 1 have a deleted, substituted, Z- or inserted amino acid sequence, and interact with human topoisomerase 2α to inhibit its activity Amino acid sequence that can be:
本発明のさらに別の態様によれば、 下記の何れかの塩基配列中の連続する 5か ら 1 0 0の塩基配列のアンチセンス配列から成るアンチセンスオリゴヌクレオチ ドが提供される。  According to still another aspect of the present invention, there is provided an antisense oligonucleotide comprising an antisense sequence of any of the following 5 to 100 base sequences in any of the following base sequences.
( a ) 配列番号 2に記載の塩基配列列；又は  (a) the nucleotide sequence set forth in SEQ ID NO: 2; or
( b ) 配列番号 2に記載の塩基配列において 1から数個の塩基が欠失、 置換及び ノ又は挿入した塩基配列を有し、 ヒ トトポイソメラーゼ 2 αと相互作用してその 活性を阻害することができるタンパク質をコードする塩基配列：  (b) having a nucleotide sequence in which one to several nucleotides have been deleted, substituted and / or inserted in the nucleotide sequence set forth in SEQ ID NO: 2, and interacting with human topoisomerase 2α to inhibit its activity; Nucleotide sequence encoding a protein capable of:
本発明のさらに別の態様によれば、 上記アンチセンスオリゴヌクレオチドを含 む、 ヒ トトポイソメラーゼ 2 ひ結合因子の阻害剤が提供される。  According to still another aspect of the present invention, there is provided an inhibitor of human topoisomerase-2 binding factor, comprising the antisense oligonucleotide.
本発明のさらに別の態様によれば、 上記アンチセンスオリゴヌクレオチドを含 む、 ヒ トトポイソメラーゼ 2 ひの発現増強剤が提供される。  According to yet another aspect of the present invention, there is provided an expression enhancer for two human topoisomerases, comprising the above antisense oligonucleotide.
本発明のさらに別の態様によれば、 上記アンチセンスオリゴヌクレオチドを含 む、 アポトーシス誘導剤が提供される。  According to still another aspect of the present invention, there is provided an apoptosis-inducing agent comprising the above antisense oligonucleotide.
本発明のさらに別の態様によれば、 上記アンチセンスオリゴヌクレオチドを含 む、 抗癌剤が提供される。  According to still another aspect of the present invention, there is provided an anticancer agent comprising the above antisense oligonucleotide.
本発明のさらに別の態様によれば、 下記の何れかのアミノ酸配列を有するヒ ト トポイソメラーゼ 2 ひ結合因子を認識する抗体、 又はその断片が提供される。 ( a ) 配列番号 1に記載のァミノ酸配列；又は  According to still another aspect of the present invention, there is provided an antibody recognizing a human topoisomerase-2 binding factor having any one of the following amino acid sequences, or a fragment thereof: (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のァミノ酸配列において 1から数個のアミノ酸が欠失、 置換及び/又は挿入したアミノ酸配列を有し、 ヒ ト トポイソメラーゼ 2 ひと相互 作用してその活性を阻害することができるァミノ酸配列：  (b) having an amino acid sequence in which one to several amino acids have been deleted, substituted, and / or inserted in the amino acid sequence of SEQ ID NO: 1 and interacting with human topoisomerase 2 to inhibit its activity Amino acid sequences that can:
本発明のさらに別の態様によれば、 被験物質の存在下において、 下記の何れか のアミノ酸配列を有するヒ トトポイソメラーゼ 2 a結合因子と、 ヒ トトポイソメ ラーゼ 2 αとの相互作用を測定し、 該相互作用を阻害する物質を選択することを 特徴とする、 ヒ トトポイソメラーゼ 2ひ結合因子の阻害剤のスクリーユング方法 が提供される。 According to still another aspect of the present invention, in the presence of a test substance, a human topoisomerase 2a binding factor having any one of the following amino acid sequences: A screening method for an inhibitor of a binding factor to human topoisomerase 2, comprising measuring an interaction with the enzyme 2α and selecting a substance that inhibits the interaction.
( a ) 配列番号 1に記載のァミノ酸配列；又は  (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のァミノ酸配列において 1から数個のァミノ酸が欠失、 置換及び Z又は挿入したアミノ酸配列を有し、 ヒ トトポイソメラーゼ 2 αと相互 作用してその活性を阻害することができるァミノ酸配列：  (b) In the amino acid sequence of SEQ ID NO: 1, one to several amino acids have a deletion, substitution, Z or insertion amino acid sequence, and interact with human topoisomerase 2α to inhibit its activity. Amino acid sequences that can be:
好ましくは、 被験物質は、 低分子化合物、 抗体、 オリゴヌクレオチド、 又はそ れらのライブラリーである。  Preferably, the test substance is a small molecule compound, an antibody, an oligonucleotide, or a library thereof.
本発明のさらに別の態様によれば、 上記方法により得られる、 ヒ トトポイソメ ラーゼ 2 α結合因子の阻害剤が提供される。 図面の簡単な説明 According to yet another aspect of the present invention, obtained by the above method, an inhibitor of human Totopoisome hydrolases 2 alpha binding agent is provided. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 ΙΤΠ aの塩基配列およびァミノ酸配列を示す。  FIG. 1 shows the base sequence and amino acid sequence of ァ a.
図 2は、 ΙΤΠ aの過剰発現による細胞内の局在を示す。  FIG. 2 shows intracellular localization due to overexpression of ΙΤΠa.
COS- 1細胞で ΙΤΠ αを発現後、 抗 Τ7抗体を用いてィムノブロットにより ΙΤΠ αの発現を確認した （図 2 (Α) )。 また、 ITI I c の過剰発現による細胞内の局在を 示した (図 2 (B) )。 COS- 1 after cell expressing Iotataupai alpha, the expression was confirmed Iotataupai alpha by Imunoburotto using anti Τ7 antibody (Fig. 2 (Alpha)). In addition, intracellular localization due to overexpression of ITI I c was shown (FIG. 2 (B)).
図 3は、 ΙΤΠ αの過剰発現によるアポトーシスの検出を示す。  FIG. 3 shows detection of apoptosis by overexpression of ΙΤΠα.
ΙΤΠ αを発現した COS- 1細胞で、 間接蛍光抗体法より PARPの切断が検出され た（図 3 (A) )。また同様に、 TUNEL法より TUNEL陽性の細胞が検出された（図 3 (B) )。  PA In COS-1 cells that expressed α, PARP cleavage was detected by indirect immunofluorescence (Fig. 3 (A)). Similarly, TUNEL-positive cells were detected by the TUNEL method (FIG. 3 (B)).
図 4は、 昆虫細胞による ΙΤΠ a発現系の構築および精製を示す。  FIG. 4 shows the construction and purification of a ΙΤΠa expression system by insect cells.
昆虫細胞による ΙΤΠ aの発現は、 BAC- T0-BAC Baculovirus Express ion Systems を用いて行った。 発現タンパク質 （矢印） を CBB 染色で確認した (A)。  Expression of ΙΤΠa by insect cells was performed using BAC-T0-BAC Baculovirus Expression Systems. The expressed protein (arrow) was confirmed by CBB staining (A).
ΙΤΠ αの精製は、 ΙΤΠ α抗体カラム （2. 5 rag の精製抗体を CNBr- activated Sepharose 4B (0. 5 g) に固定化して調製） を用いて行った。 溶出した各フラク シヨン （1〜8) は透析後、 SDS- PAGE、 銀染色を行ってタンパク質の精製を確認し た (B)。 Purification of ΙΤΠα was performed using a ΙΤΠα antibody column (prepared by immobilizing 2.5 rag of purified antibody on CNBr-activated Sepharose 4B (0.5 g)). The eluted fractions (1-8) were dialyzed and subjected to SDS-PAGE and silver staining to confirm protein purification. (B).
図 5は、 組み換え ΙΤΠ αによる Τ0ΡΠ a活性の阻害作用を示す。  FIG. 5 shows the inhibitory effect of recombinant ΙΤΠα on Τ0ΡΠa activity.
昆虫細胞、 バキュロウィルスを用いて発現させた精製 ΙΤΠα (図 4、 フラクシ ヨン 3) 力 ΤΟΡΠαの活性 （Relaxation, Decatenation) に対して阻害作用を 示すかどうか検討した。 （A) Decatenation 活性の測定。 基質にキネトプラス ト DNA (0.175^g/assay) を用いた。 (B) Relaxation活性の測定。基質に PUC19 (0.3 μ g/assay) ·≥：用レヽた 0 Purified ΙΤΠα expressed in insect cells and baculovirus (Fig. 4, fraction 3) The ability to inhibit the activity of ΤΟΡΠα (Relaxation, Decatenation) was examined. (A) Measurement of Decatenation activity. Kinetoplast DNA (0.175 ^ g / assay) was used as a substrate. (B) Measurement of Relaxation activity. P UC19 (0.3 μg / assay) for the substrate · ≥: 0
図 6は、 細胞周期における ΙΤΠαの発現量の変化と細胞内の局在を示す。 正常ヒ ト皮膚線維芽細胞 （NB1-RGB) を 72 時間低血清培地 （0.4%FBS, non-essential amino acids を含む D- MEM) で培養し、 その後、 15%FBSになるよ うに血清を添加した。 この時点を 0時間として 4時間おきに 32時間まで細胞を 回収し、 レーザースキャニングサイ トメ一ターによる DNAの含有量をヒストグラ ム、および G1/G0，S,G2/M期の含有率を％で示した（A)。ィムノブ口ットにより ΙΤΠ と T0PI のタンパク質の発現、 および NIH Imageを用いてその発現量を測定 した（B)。 8時間後から 4時間おきに 28時間まで ΙΤΠαと Τ0ΡΙΙ αの細胞内の局 在を検出した（0。 血清添加 32時間後の細胞を回収し、 細胞質およぴ核分画を以 下の方法で調製した。 細胞を PBS (-)で 2回洗浄し， 400 /z lの溶液 A (360 μΐの Nuclear isolation buffer (NIB) に 40/ 1 の 10% (v/v) Triton X- 100を添加） に縣濁した。 氷中に 5分間静置後， 遠心 （1000 gで 90秒） した。 上清液を細胞 質分画とし， 沈澱物は 360 iの溶液 B (0.35 M NaClを含む NIB (30 mM Tris-HCl (pH 7.5) , 1.5mM MgCl2, 10 raM KC1, 20% (v/v) glycerol)) に縣濁して， 氷中 に 30分間静置した。その後， 遠心（10，000 gX5分） し、上清液を核分画とした。 ▲が Τ0ΡΠα、 △が ΙΤΠαのバンドを示す。 また， 抗 ΙΤΠ α抗体の抗原として使 用したべプチドを過剰量添加して， 両分画のバンドが ΙΤΠ aに相当することを検 討した（D)。 FIG. 6 shows changes in the expression level of ΙΤΠα in the cell cycle and localization within the cell. Normal human dermal fibroblasts (NB1-RGB) are cultured in a low serum medium (D-MEM containing 0.4% FBS, non-essential amino acids) for 72 hours, and then serum is added to 15% FBS. did. The cells were collected every 4 hours up to 32 hours, taking this time as 0 hour, and the DNA content by laser scanning cytometer was determined by histogram and the G1 / G0, S, G2 / M phase content was calculated as%. (A). The expression of the ΙΤΠ and T0PI proteins was measured using a simulated knob and their expression levels were measured using NIH Image (B). 8α and Τ0ΡΙΙα subcellular localization was detected from 8 hours to 4 hours every 28 hours (0. Cells were collected 32 hours after addition of serum, and cytoplasmic and nuclear fractions were determined as follows. Wash the cells twice with PBS (-), and add 40/1 10% (v / v) Triton X-100 to 400 μz solution A (360 μΐ Nuclear isolation buffer (NIB)) After centrifugation at 1000 g for 90 seconds, the supernatant was used as the cytoplasmic fraction, and the precipitate was 360i solution B (NIB containing 0.35 M NaCl). (30 mM Tris-HCl (pH 7.5), 1.5 mM MgCl 2 , 10 raM KC1, 20% (v / v) glycerol)), and left to stand on ice for 30 minutes. 000 g X5 min), and the supernatant was used as the nuclear fraction. ▲ indicates the band of Τ0ΡΠα, △ indicates the band of ΙΤΠα.Add the excess amount of the peptide used as the antigen of the anti-ΙΤΠα antibody. Consider that both fraction bands correspond to ΙΤΠa And (D).
図 7は、 抗 ΙΤΠα抗体カラムを用いた ΙΤΠαと ΤΟΡΠαの相互作用の検討を示 す。 ヒ ト皮膚線維芽細胞 （1X108細胞） を超音波破砕後、 超遠心を行い、 その上清 液を抗 ΙΤΠ α抗体一ァフィ二ティ一カラムに流した。 洗浄後、 TritonX- 100を含 んだ 50 mMグリシン溶液 （pH2.5) で溶出して直ちに中和後、 透析した。 その後、 サンプルは、 抗 ΙΤΠα抗体および抗 ΤΟΡΠα抗体を用いてィムノブロットを行つ た。 FIG. 7 shows the examination of the interaction between ΙΤΠα and ΤΟΡΠα using an anti-ΙΤΠα antibody column. After sonication of human skin fibroblasts (1 × 10 8 cells), ultracentrifugation was performed, and the supernatant was applied to an anti-αα antibody affinity column. After washing, the mixture was eluted with a 50 mM glycine solution (pH 2.5) containing TritonX-100, immediately neutralized, and dialyzed. Thereafter, the sample was subjected to immunoblot using an anti-α antibody and an anti-α antibody.
図 8は、プラズモン共鳴法を用いた ΊΤΠαと ΤΟΡΠαの相互作用の検討を示す。 プラズモン共鳴法による測定は、 IAsys plusを用いて行った。 固相化用のキュ ベットには CMデキス トランキュベットを使用して、 ΙΤΠ αを固相化 （267 arc seconds) した。 T0PII αを 2.9〜22.2ηΜの範囲内で添加し、このデータを FASTfit にて解析し、 会合速度定数 （kass：)、 解離速度定数 （kdiss) および解離平行定数 を算出した。  FIG. 8 shows a study of the interaction between ΊΤΠα and ΤΟΡΠα using plasmon resonance. The measurement by the plasmon resonance method was performed using IAsys plus. ΙΤΠα was immobilized (267 arc seconds) using a CM dextran cuvette as a cuvette for immobilization. T0PIIα was added within the range of 2.9 to 22.2ηΜ, and this data was analyzed by FASTfit to calculate the association rate constant (kass :), dissociation rate constant (kdiss) and dissociation parallel constant.
図 9は、 アンチセンスオリゴヌクレオチドによる ΤΟΡΠ αの発現量およびその 局在を示す。  FIG. 9 shows the expression amount and localization of ΤΟΡΠα by the antisense oligonucleotide.
正常ヒ ト皮膚線維芽細胞 （1X106細胞/ ml) にセンス、 センスリバース、 ラン ダムおよびアンチセンスオリゴヌクレオチドを導入し、 48 時間後、 抗 ΙΤΠα抗 体を用いてィムノブロットを行い、 細胞内の ΙΤΠαの発現量を測定した。 また、 アンチセンスオリゴヌクレオチドの濃度をかえて ΙΤΠαの発現量の変動を測定 した。 さらに、 アンチセンスオリゴヌクレオチドを導入後、 細胞内の ΙΤΙΙαおよ ぴ ΤΟΡΠαの発現量を経時的に測定し、 アンチセンスオリゴヌクレオチドによる T0PII aの局在の変化は間接傾向抗体法を用いて検出した。 Sense, sense reverse, random and antisense oligonucleotides were introduced into normal human skin fibroblasts (1 × 10 6 cells / ml), and 48 hours later, immunoblotting was performed using an anti-αα antibody, and intracellular ΙΤΠα was detected. Was measured. In addition, changes in the expression level of ΙΤΠα were measured by changing the concentration of the antisense oligonucleotide. Furthermore, after introducing the antisense oligonucleotide, the expression levels of ΙΤΙΙα and ぴ α in the cells were measured over time, and changes in the localization of T0PIIa by the antisense oligonucleotide were detected using the indirect tendency antibody method. .
図 1 0は、 正常細胞 （ヒ ト皮膚線維芽細胞） とがん細胞 （Jurcat、 HL60、 HeLa、 腎癌、 直腸癌、 胆嚢癌、 大腸癌） での ΙΤΠαと Τ0ΡΙΙαの発現量の比較を示す。 Figure 1 0 is, normal cells (human dermal fibroblasts) and cancer cells (Jurcat, HL60, HeLa, renal cancer, rectal cancer, gallbladder cancer, colon cancer) comparisons ΙΤΠα and Tau0roiotaiota alpha expression levels in Show.
ΙΤΠαの発現量は、 がん細胞では正常細胞に比べて、 2〜3倍減少し、 ΤΟΡΙΙα は、 逆に 2〜3倍増加した （表 1)。  The expression level of ΙΤΠα decreased 2-3 times in cancer cells compared to normal cells, and ΤΟΡΙΙα increased 2-3 times in cancer cells (Table 1).
図 1 1は、 アンチセンスオリゴヌクレオチドの正常細胞と癌細胞に対する作用 効果を示す。  FIG. 11 shows the effect of the antisense oligonucleotide on normal cells and cancer cells.
正常細胞 （NB1- RGB) と癌細胞 （HeLa) に対して 2/xM のアンチセンスをリポ フエクチン法で導入した。 48時間後、 LSCで解析した（A)。アンチセンス導入 24、 48、 69時間後、 正常細胞と癌細胞を回収して DNAの ladderを検出した（B)。 さら に、 アンチセンス導入 48時間後の癌細胞について、 間接蛍光抗体法により、 PARP の切断の有無を測定した（0。 Liposome 2 / xM antisense against normal cells (NB1-RGB) and cancer cells (HeLa) Introduced by the pectin method. After 48 hours, analysis was performed by LSC (A). At 24, 48, and 69 hours after the introduction of antisense, normal cells and cancer cells were recovered and DNA ladders were detected (B). Furthermore, the presence or absence of PARP cleavage was measured by indirect immunofluorescence on cancer cells 48 hours after transfection with antisense (0).
図 1 2は、 ΙΤΠ αの合成ペプチドによる ΤΟΡΠ αの阻害作用の検出を示す。 ΙΤΙΙ αのァミノ酸配列 132〜151 (VTATFPYTTILSIWLATRRV)が濃度依存的に Τ0ΡΙΙ α活性を阻害した。 Re 1 axa t i on活性については、 20 Mでほぼ完全に阻害した（ A)。 Decatenation活性については、 100 μ Mでも完全に阻害しなかった（Β)ことから、 両活性に対して感受性の違いが認められた。 発明を実施するための最良の形態  FIG. 12 shows detection of the inhibitory effect of 合成 α by the synthetic peptide of ΙΤΠα. The amino acid sequence 132-151 of ΙΤΙΙα (VTATFPYTTILSIWLATRRV) inhibited Τ0ΡΙΙα activity in a concentration-dependent manner. Re 1 axation activity was almost completely inhibited at 20 M (A). Decatenation activity was not completely inhibited even at 100 μM (Β), indicating a difference in sensitivity to both activities. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明の実施の形態について詳細に説明する。  Hereinafter, embodiments of the present invention will be described in detail.
( 1 ) ΙΤΠ α及びそれを含む薬剤  (1) ΙΤΠα and drugs containing it
本発明は、 ΙΤΠ αを有効成分として含む薬剤に関するもので、 該薬剤は、 Τ0ΡΠ α阻害剤又はアポトーシス誘導剤として使用できる。 本発明で用いる ITII c は、 以下の何れかのァミノ酸を有する。  The present invention relates to a drug containing ΙΤΠα as an active ingredient, and the drug can be used as a Τ0ΡΠα inhibitor or an apoptosis inducer. ITII c used in the present invention has any of the following amino acids.
( a ) 配列番号 1に記載のァミノ酸配列；又は  (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のァミノ酸配列において 1から数個のァミノ酸が欠失、 置換及び/又は挿入したアミノ酸配列を有し、 Τ0ΡΠ αと相互作用してその活性 を阻害することができるアミノ酸配列：  (b) one or several amino acids in the amino acid sequence described in SEQ ID NO: 1 have a deleted, substituted and / or inserted amino acid sequence, and may interact with Τ0ΡΠα to inhibit its activity. Possible amino acid sequences:
本明細書において、 「配列番号 1に記載のアミノ酸配列において 1から数個の アミノ酸が欠失、置換及ぴノ又は挿入したアミノ酸配列」 における 「1から数個」 の範囲は特には限定されないが、 例えば、 1から 2 0個、 好ましくは 1から 1 0 個、 より好ましくは 1から 7個、 さらに好ましくは 1から 5個、 特に好ましくは 1から 3個程度を意味する。  In the present specification, the range of `` 1 to several '' in the `` amino acid sequence in which one to several amino acids have been deleted, substituted and substituted or inserted in the amino acid sequence of SEQ ID NO: 1 '' is not particularly limited. For example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, particularly preferably about 1 to 3.
本明細書において 「Τ0ΡΙΙ αと相互作用してその活性を阻害することができる アミノ酸配列」 とは、 該アミノ酸配列を有するタンパク質が、 配列番号 1に記載 のアミノ酸配列を有するタンパク質と同程度又はそれ以上の親和性で ΤΟΡΙΙ αと 相互作用してその活性が阻害されることを意味する。 Τ0ΡΠ aの活性の阻害は、 適当な方法でァッセィすることができる。 As used herein, “an amino acid sequence capable of interacting with {0} α to inhibit its activity” refers to a protein having the amino acid sequence described in SEQ ID NO: 1. Means that the protein interacts with ΤΟΡΙΙα with the same or higher affinity as a protein having the amino acid sequence of α to inhibit its activity. Inhibition of {0} a activity can be assayed by any appropriate method.
ΤΟΡΠ αの活性の阻害は、 例えば、 ΤΟΡΠ αの Relaxation活性及び Decatenation 活性に対する阻害作用を測定することにより評価することができる。 例えば、 Decatenation活性の測定には、 基質にキネトプラスト D N Aを用い、 Relaxation 活性の測定には、 基質にスーパーコィリング D N Aを用いてアツセィを行うこと ができる。 活性に対する阻害効果は、 例えば、 ΙΤΠ αを加えなかった時の反応後 の D N A量を 1 0 0 %として ΙΤΠ αを加えた時の D N A量を相対的に算出する ことにより評価することができる。  The inhibition of the activity of で き る α can be evaluated, for example, by measuring the inhibitory effect of ΤΟΡΠα on Relaxation activity and Decatenation activity. For example, for the determination of Decatenation activity, kinetoplast DNA can be used as the substrate, and for the measurement of the Relaxation activity, the assay can be performed using Supercoiling DNA as the substrate. The inhibitory effect on the activity can be evaluated by, for example, assuming that the amount of DNA after the reaction without adding αα is 100%, and calculating the amount of DNA when adding Δα relatively.
本発明では、 配列番号 1に記載のァミノ酸配列における部分ァミノ酸配列を有 するタンパク質を、 ΤΟΡΙΙ α阻害剤として使用することができる。 部分アミノ酸 配列の長さは特に限定されず、例えば、 5アミノ酸残基から 1 0 0アミノ酸残基、 好ましくは 5アミノ酸残基から 5 0アミノ酸残基、 さらに好ましくは、 5ァミノ 酸残基から 3 0アミノ酸残基、 特に好ましくは 5アミノ酸残基から 2 0アミノ酸 残基程度である。 そのような部分アミノ酸配列を有するタンパク質としては、 例 えば、 配列番号 1 0に記載のアミノ酸配列を有するタンパク質が挙げられる。 こ のような部分アミノ酸配列を有するタンパク質は、 ペプチド合成機を用いた通常 のぺプチド合成法により作製することができる。  In the present invention, a protein having a partial amino acid sequence in the amino acid sequence of SEQ ID NO: 1 can be used as a ΤΟΡΙΙα inhibitor. The length of the partial amino acid sequence is not particularly limited, and is, for example, from 5 amino acid residues to 100 amino acid residues, preferably from 5 amino acid residues to 50 amino acid residues, and more preferably from 5 amino acid residues to 3 amino acid residues. It is about 0 amino acid residues, particularly preferably about 5 to 20 amino acid residues. As a protein having such a partial amino acid sequence, for example, a protein having the amino acid sequence of SEQ ID NO: 10 can be mentioned. A protein having such a partial amino acid sequence can be produced by a normal peptide synthesis method using a peptide synthesizer.
次に、 配列番号 1に記載のァミノ酸配列を有するタンパク質の入手 ·製造方法 について説明する。 配列番号 1に記載のアミノ酸配列を有するタンパク質の入 手 ·製造方法は特に限定されず、 天然由来のタンパク質でも、 化学合成したタン パク質でも、 遺伝子組み換え技術により作製した組み換えタンパク質の何れでも よい。 比較的容易な操作でかつ大量に製造できるという点では、 組み換えタンパ ク質が好ましい。  Next, a method for obtaining and producing a protein having the amino acid sequence described in SEQ ID NO: 1 will be described. The method for obtaining the protein having the amino acid sequence of SEQ ID NO: 1 is not particularly limited, and may be a naturally occurring protein, a chemically synthesized protein, or a recombinant protein produced by a genetic recombination technique. Recombinant proteins are preferred because they are relatively easy to operate and can be produced in large quantities.
配列番号 1に記載のァミノ酸配列を有するタンパク質を製造するには、 該タン パク質をコードする塩基配列 （例えば、 配列番号 2に記載の塩基配列） を有する DNAを作製し、 これを好適な発現系に導入することにより目的タンパク質を製 造することができる。 In order to produce a protein having the amino acid sequence of SEQ ID NO: 1, a protein having the nucleotide sequence encoding the protein (for example, the nucleotide sequence of SEQ ID NO: 2) A target protein can be produced by preparing DNA and introducing it into a suitable expression system.
配列番号 2に記載の塩基配列を有する DNAは、 ヒ ト由来 （例えば、 HeLa細胞 由来など） の cDN Aライブラリーを、 配列番号 2に記載の塩基配列の情報に基 づいて設計した好適なプライマ一又はプローブを用いてスクリーユングすること により入手できる。 スクリーニングはプラークハイブリダィゼーシヨン等で行う ことができる。 あるいは、 ヒ ト由来 （例えば、 HeLa細胞由来など） の c DNAラ ィブラリーを铸型として使用し、 配列番号 2に記載の塩基配列の情報に基づいて 設計した好適なプライマ一を用いて PCRを行うことにより、 目的遺伝子を直接 クローニングすることもできる。  The DNA having the nucleotide sequence of SEQ ID NO: 2 is a suitable primer designed based on the information of the nucleotide sequence of SEQ ID NO: 2 from a human-derived (for example, HeLa cell-derived) cDNA library. It can be obtained by screening using one or a probe. Screening can be performed by plaque hybridization or the like. Alternatively, PCR is performed using a human-derived cDNA library (for example, derived from HeLa cells) as type III and using a suitable primer designed based on the nucleotide sequence information set forth in SEQ ID NO: 2. This allows direct cloning of the target gene.
組み換えタンパク質を発現させるための発現系 （遺伝子を含む発現ベクターと その宿主） は当業者に公知である。  Expression systems (expression vectors containing genes and their hosts) for expressing the recombinant protein are known to those skilled in the art.
DNAを宿主細胞中で発現させるためには、 まず、 該 DNAを発現ベクター中 のプロモーターの下流に挿入し、 次いでこの組み換え発現ベクターを、 当該発現 ベクターに適合した宿主細胞中に導入する。  In order to express DNA in a host cell, first, the DNA is inserted downstream of a promoter in an expression vector, and then the recombinant expression vector is introduced into a host cell suitable for the expression vector.
細菌用の発現ベクターとしては、 pGEMEX— 1 (P r ome g a社製）、 p QE- 9 (Q I AG EN社製）、 p QE- 30 (Q I AG EN社製）、 pRSET Examples of expression vectors for bacteria include pGEMEX-1 (produced by Promega), p QE-9 (produced by QI AG EN), p QE-30 (produced by Q I AG EN), pRSET
( I n v i t r o g e n社製）、 p LEX ( I n v i t r o g e n社製）、 p T r cH i s ( I n v i t r o g e n社製）、 pGEX (Ph a rma c i a社製）、 p ET (No v a g e n社製）などが挙げられ、酵母用の発現ベクターとしては、 例えば、 YE p 13 (ATCC 371 1 5)、 YEp 24 (ATCC 37051)、 Y c p 5 O (ATCC 374 1 9), pHS 19、 p HS 1 5等が挙げられ、 バキ ュロ用の発現ベクターとしては、 p F a s t B a c (G i b c o BRL社製）、 p VL 1 392 ( I n v i t r o g e n社製） 等が挙げられ、 動物細胞用の発現べ クタ一として、 例えば、 p c DNA I、 p c DM 8 (フナコシ社）、 p c DNA I /AmP ( I n v i t r o g e n社製）、 p RE P 4 ( I n v i t r o g e n社製） などや、 組換えウィルス作成用発現ベクター、 例えば、 pMFG (Ta k a r a 社製）、 pAd e x (Ta k a r a社製） などが挙げられる。 (Invitrogen), pLEX (Invitrogen), pTrcHis (Invitrogen), pGEX (Pharmacia), pET (Novagen) and the like. Examples of expression vectors for yeast include, for example, YE p13 (ATCC 371 15), YEp 24 (ATCC 37051), Ycp5O (ATCC 374 19), pHS 19, pHS 15 and the like. Examples of expression vectors for baculo include pFastBac (manufactured by Gibco BRL), pVL1392 (manufactured by Invitrogen), and the like. , PcDNAI, pcDM8 (Funakoshi), pcDNAI / AmP (Invitrogen), pREP4 (Invitrogen), and expression vectors for producing recombinant viruses, such as pMFG ( Ta kara And pAd ex (Takara).
細菌用の発現ベクターに用いることができるプロモーターとしては、 例えば、 t r pプロモーター （P t r p)、 T7プロモータ一、 1 a cプロモーター （P l a c), PLプロモーター、 PRプロモーター、 PSEプロモーター等の大腸菌や ファージ等に由来するプロモーター等を挙げることができる。 酵母用の発現べク ターに用いることができるプロモータ一としては、 例えば、 PH05プロモータ 一、 PGKプロモーター、 GAPプロモーター、 ADHプロモーター、 g a l l プロモーター、 g a l 10プロモーター、 ヒートショックタンハ°ク質プロモータ 一、 MF a lプロモータ一、 CUP 1プロモーター等を挙げることができる。 バ キュロ用の発現ベクターに用いることができるプロモーターとしては、 例えば、 ポリヘドリンプロモータ一等を挙げることができる。 動物細胞用の発現ベクター に用いることができるプロモーターとしては、例えば、サイ トメガロウィルス（ヒ ト CMV)の I E (immediate early)遺伝子のプロモーター、 S V40の初期プロ モーター、 レトロウイノレスのプロモータ一、 アデノウイノレスのプロモーター、 メ タロチォネインプロモーター、 ヒートショックプロモーター、 SRaプロモータ 一、 ァクチンプロモーター等を挙げることができる。 また、 ヒ ト CMVの I E遺 伝子のェンハンサーをプロモーターと共に用いてもよい。 Promoters which can be used in expression vectors for bacterial, e.g., trp promoter (P trp), T7 promoter one, 1 ac promoter (P lac), P L promoter, P R promoter, Ya E. coli, such as P SE promoter Examples include a phage-derived promoter and the like. Examples of promoters that can be used in yeast expression vectors include PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gall promoter, gal10 promoter, heat shock protein promoter, and MF. al promoter, CUP 1 promoter and the like. Examples of a promoter that can be used in an expression vector for baculo include polyhedrin promoter and the like. Examples of a promoter that can be used in an expression vector for animal cells include the promoter of the immediate early (IE) gene of the cytomegalovirus (human CMV), the early promoter of SV40, the promoter of retrowinolas, and the adeno-adenovirus. Winores promoter, metallothionein promoter, heat shock promoter, SRa promoter, actin promoter and the like. Further, the enhancer of IE gene of human CMV may be used together with the promoter.
宿主細胞としては、 目的タンパク質を発現できるものであれば特に制限されず、 細菌、 酵母、 動物細胞、 昆虫細胞などが挙げられる。 より具体的には、 エツシェ リヒア属、 セラチア属、 コリネバクテリウム属、 ブレビバクテリウム属、 シュ一 ドモナス属、 バチルス属、 ミクロバクテリゥム属等に属する細菌、 クルイべロミ セス属、 サッカロマイセス属、 シゾサッカロマイセス属、 トリコスポロン属、 シ ヮニォミセス属等に属する酵母、 ナマルバ細胞、 He L a細胞 COS 1細胞、 C OS 7細胞、 CHO細胞、 293細胞などの動物細胞、 S f 9、 S f 21、 H i F i v eなどの昆虫細胞等を挙げることができる。 The host cell is not particularly limited as long as it can express the target protein, and includes bacteria, yeast, animal cells, insect cells, and the like. More specifically, bacteria belonging to the genus Etscherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., Kluyveromyces, Saccharomyces, Yeasts belonging to the genus Schizosaccharomyces, Trichosporon, Genus Schiziomyces, Namalba cells, HeLa cells COS 1 cells, COS 7 cells, CHO cells, animal cells such as 293 cells, Sf9, Sf21, And insect cells such as Hive.
組換えベクターの宿主への導入方法は、 例えば、 リン酸カルシウム法、 プロト プラスト法、エレク トロポレーシヨン法、スフ：！:ロブラスト法、酢酸リチウム法、 リポフエクション法などが挙げられ、 宿主細胞の種類に応じて適宜選択すること ができる。 Methods for introducing a recombinant vector into a host include, for example, a calcium phosphate method, a protoplast method, an electroporation method, and a suffix :! : Roblast method, lithium acetate method, A lipofection method and the like can be mentioned, and it can be appropriately selected according to the type of the host cell.
本発明では、 バキュロウィルスを用いて昆虫細胞で発現させた組み換えタンパ ク質を使用することが好ましい。  In the present invention, it is preferable to use a recombinant protein expressed in insect cells using a baculovirus.
昆虫細胞を宿主として用いる場合には、 組換え遺伝子導入ベクターおよびバキ ュロウィルスを昆虫細胞に共導入して昆虫細胞培養上清中に組換えウィルスを得 た後、 さらに組換えウィルスを昆虫細胞に感染させ、 タンパク質を発現させるこ と刀 できる (例 は、 Baculovirus Expression Vectors, A Laboratory Manual ； 及びカレン ト · プロ ト コールズ . イン ' モレキュラー ' バイオロジー、 Bio/Technology, 6, 47 (1988)等に記載）。  When an insect cell is used as a host, the recombinant gene transfer vector and baculovirus are co-transfected into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is transmitted to the insect cells. (Examples are described in Baculovirus Expression Vectors, A Laboratory Manual; and Current Protocols in 'Molecular' biology, Bio / Technology, 6, 47 (1988), etc.). ).
バキュロウィルスとしては、 例えば、 ョ トウガ科昆虫に感染するウィルスであ るアウ トグラファ ' カリフォルニ力 ·ヌクレアー · ポリへドロシス · ウィルス (Autographs californica nuclear polyhedrosis virus)等 ¾用レヽること力 で、き る。  The baculovirus can be obtained, for example, by the use of Autographs californica nuclear polyhedrosis virus, which is a virus that infects Aspergillus insects, such as Autographi californica, nucleus, polyhedrosis virus, and the like.
昆虫細胞としては、 Spodoptera frugiperdaの卵巣細胞である S f 9、 S f 2 1 〔バキュロウィルス 'エクスプレッション .ベクターズ、 ァ ' ラボラ トリー · マニュアル、ダブリユー 'ェイチ 'フリーマン 'アンド'カンパニー（W. H. Freeman and Company) , ニューヨーク（New York)、 （1992)〕、 Trichoplusia ni の卵巣細 胞である H i F i v e (インビトロジェン社製）等を用いることができる。  As insect cells, Sf9, Sf21, ovarian cells of Spodoptera frugiperda [Baculovirus 'Expression. Vectors, A' Laboratories Manual, Doublet 'Jay' Freeman 'and' Company (WH Freeman and Company) New York, (1992)], and Trichoplusia ni ovarian cells, Hi Five (manufactured by Invitrogen Corporation) and the like can be used.
組換えウィルスを調製するための、 昆虫細胞への組換え遺伝子導入ベクターと 上記バキュロウィルスの共導入方法としては、 例えば、 リン酸カルシウム法又は リポフエクシヨン法等を挙げることができる。  Examples of a method of co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include a calcium phosphate method and a Lipofexion method.
上記のようにして作製した目的 D N Aを有する組み換え発現べクタ一を保有す る形質転換体を培地に培養し、 培養物中に目的タンパク質を生成蓄積させ、 該培 養物より目的タンパク質を採取することにより、 組み換えタンパク質を単離する ことができる。  The transformant having the recombinant expression vector having the target DNA prepared as described above is cultured in a medium, the target protein is produced and accumulated in the culture, and the target protein is collected from the culture. Thereby, the recombinant protein can be isolated.
形質転換体の培養物から、 目的の組み換えタンパク質を単離精製するには、 通 常のタンパク質の単離、精製法を用いればよレ、。例えば、組み換えタンパク質が、 細胞内に溶解状態で発現した場合には、 培養終了後、 細胞を遠心分離により回収 し水系緩衝液に懸濁後、 超音波破砕機、 フレンチプレス、 マントンガウリンホモ ゲナイザー、 ダイノミル等により細胞を破碎し、 無細胞抽出液を得る。 該無細胞 抽出液を遠心分離することにより得られた上清から、 通常のタンパク質の単離精 製法、即ち、溶媒抽出法、硫安等による塩析法、脱塩法、有機溶媒による沈殿法、 ジェチルアミノエチル（DEAE)セファロース、 DIAION HPA-75 (三菱化成社製）等レ ジンを用いた陰イオン交換クロマトグラフィー法、 S-Sepharose FF (フアルマシ ァ社製）等のレジンを用いた陽イオン交換クロマトグラフィ一法、 プチルセファ ロース、フエ二ルセファロース等のレジンを用いた疎水性クロマトグラフィ一法、 分子篩を用いたゲルろ過法、 ァフィ二ティークロマトグラフィー法、 クロマトフ オーカシング法、 等電点電気泳動等の電気泳動法等の手法を単独あるいは組み合 わせて用い、 精製標品を得ることができる。 To isolate and purify the target recombinant protein from a transformant culture, You can use the usual protein isolation and purification methods. For example, when the recombinant protein is expressed in a lysed state in the cells, the cells are collected by centrifugation after cell culture, suspended in an aqueous buffer, and then sonicated, a French press, a Mentongaulin homogenizer, and the like. The cells are disrupted using a dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal protein isolation and purification method, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resins such as getylaminoethyl (DEAE) Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Kasei), and cations using resins such as S-Sepharose FF (manufactured by Pharmacia) Exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose, phenylsepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing, etc. A purified sample can be obtained using a technique such as electrophoresis alone or in combination.
配列番号 1に記載のアミノ酸配列において 1から数個のアミノ酸が欠失、 置換 及び Z又は挿入したアミノ酸配列を有するタンパク質は、 配列番号 1に記載のァ ミノ酸配列及び配列番号 2に記載の塩基配列の情報に基づいて当業者であれば適 宜製造することができる。例えば、化学合成、 P C R等を含む遺伝子工学的手法、 突然変異誘発などの当業者に既知の任意の方法で、 作製することもできる。 具体 的には、 配列番号 2に記載の塩基配列を有する D N Aを利用し、 これら D N Aに 変異を導入することにより変異 D N Aを取得することができる。  A protein having the amino acid sequence of SEQ ID NO: 1 in which one to several amino acids have been deleted, substituted, Z- or inserted has an amino acid sequence represented by SEQ ID NO: 1 and a base sequence represented by SEQ ID NO: 2. Those skilled in the art can appropriately produce the protein based on the sequence information. For example, it can be produced by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques including PCR, mutagenesis, and the like. Specifically, a mutant DNA can be obtained by using a DNA having the nucleotide sequence of SEQ ID NO: 2 and introducing a mutation into these DNAs.
例えば、 配列番号 2に記載の塩基配列を有する D N Aに対し、 変異原となる薬 剤と接触作用させる方法、 紫外線を照射する方法、 遺伝子工学的手法等を用いて 行うことができる。  For example, the method can be carried out using a method in which DNA having the base sequence of SEQ ID NO: 2 is brought into contact with a drug as a mutagen, a method of irradiating ultraviolet rays, a genetic engineering technique, or the like.
遺伝子工学的手法の一つである部位特異的変異誘発法は特定の位置に特定の変 異を導入できる手法であることから有用であり、 モレキュラークローユング第 2 版、力レント 'プロ トコールズ.イン'モレキュラー 'バイオロジー、 Nucleic Acids Research, 10, 6487, 1982、 Nucleic Acids Research, 12, 9441, 1984、 Nucleic Acids Research, 13, 4431, 1985、 Nucleic Acids Research, 13, 8749, 1985、 Proc. Natl. Acad. Sci. USA, 79, 6409, 1982、 Proc. Natl. Acad. Sci. USA, 82, 488, 1985、 Gene, 34, 315, 1985、 Gene, 102, 67, 1991等に記載の方法に 準じて行うことができる。 Site-directed mutagenesis, one of the genetic engineering techniques, is useful because it is a technique that can introduce a specific mutation at a specific position, and is useful in Molecular Cloning, 2nd edition, Ryrent's Protocols. 'Molecular' biology, Nucleic Acids Research, 10, 6487, 1982, Nucleic Acids Research, 12, 9441, 1984, Nucleic Acids Research, 13, 4431, 1985, Nucleic Acids Research, 13, 8749, 1985, Proc. Natl. Acad. Sci. USA, 79, 6409, 1982, Proc. Natl. Acad. Sci. USA, 82, 488, 1985. Gene, 34, 315, 1985, Gene, 102, 67, 1991, and the like.
上記した方法により、 配列番号 2に記載の塩基配列において変異を有する塩基 配列を有する D N Aを入手し、 この D N Aを上記と同様に発現させることにより 、 目的タンパク質を製造することができる。  The target protein can be produced by obtaining DNA having a nucleotide sequence having a mutation in the nucleotide sequence of SEQ ID NO: 2 by the above-described method, and expressing the DNA in the same manner as described above.
本発明の薬剤は、 ΤΟΡΠ α阻害剤として使用でき、 その具体的用途は特に限定 されないが、 例えば、 アポトーシス誘導剤として使用することができる。  The agent of the present invention can be used as a ΤΟΡΠα inhibitor, and its specific use is not particularly limited. For example, it can be used as an apoptosis inducer.
アポトーシス（apoptosis)は古典的細胞死である壊死（ネクローシス、 necrosis) とは形態学的に異なる細胞死として最初に発見され定義され、 その後の研究から アポトーシスの誘導および抑制は遺伝子によって支配される、 いわゆるプロダラ ムされた細胞死であることが判ってきた。アポトーシスでは細胞の活性化に伴レ、、 複雑な生化学反応が起こり、 種々のタンパク質や DNAの分解酵素が産生され、 こ れが自身の細胞に作用して細胞死がもたらされる。 アポトーシスは正常な発生 · 分化に不可欠な生理的細胞死であり、 正常な生体組織の細胞回転などにおいて 個々の細胞に起こっている。 そのため、 アポトーシスが過剰に減少すると多くの 機能障害の原因になることが判明している。 例えば、 アポトーシスの減少に起因 する疾患としては悪性腫瘍（癌）、 白血病、 自己免疫性疾患、 ウィルス感染疾患（H I V感染など）、 増殖性皮膚疾患、 慢性関節リウマチ、 自己免疫疾患、 肝炎、 腎疾 患等を挙げることができる。 従って、 本発明のアポトーシス誘導剤は、 これらの アポトーシスの減少に起因する疾患の治療及び Z又は予防剤として使用すること ができる。  Apoptosis was first discovered and defined as morphologically distinct cell death from the classical cell death, necrosis, and subsequent studies have shown that induction and suppression of apoptosis are governed by genes. It has turned out to be so-called programmed cell death. In apoptosis, a complex biochemical reaction occurs along with the activation of cells, producing various protein and DNA degrading enzymes, which act on their own cells to cause cell death. Apoptosis is a physiological cell death that is essential for normal development and differentiation, and occurs in individual cells during normal biological tissue cell rotation. Therefore, it has been shown that excessive reduction of apoptosis causes many dysfunctions. For example, diseases caused by decreased apoptosis include malignant tumors (cancer), leukemias, autoimmune diseases, viral infectious diseases (such as HIV infection), proliferative skin diseases, rheumatoid arthritis, autoimmune diseases, hepatitis, and kidney diseases. And the like. Therefore, the apoptosis-inducing agent of the present invention can be used as a therapeutic or Z- or prophylactic agent for these diseases caused by a decrease in apoptosis.
アポトーシスの形態学的特徴として、 周囲の細胞との接触の欠乏、 細胞質の濃 縮化、 エンドヌクレアーゼの活性に関連したクロマチンの凝縮及び核凝縮、 核の 分節化等を挙げることができ、 更に、 細胞表面の微絨毛の消失及び細胞表面の平 滑化 （細胞表面の水泡形成： membraneblebbing ) 等も観察される。 また、 ェン ドヌクレアーゼ活性により、 DNAが断片化する現象も観察され、 細胞自体がァ ポト一シス小体とよばれる細胞断片を形成し、 この形成されたアポトーシス小体 力 迅速に周囲の細胞やマクロファージ等により貪食分解され、 アポトーシスが 起こるとされている。 従って、 アポトーシスの確認は、 例えば、 細胞から抽出し た DNAの断片化と細胞の形態的な観察などで行うことができる。 Morphological features of apoptosis include lack of contact with surrounding cells, cytoplasmic enrichment, chromatin condensation and nucleus condensation associated with endonuclease activity, and nuclear segmentation. Disappearance of microvilli on the cell surface and smoothing of the cell surface (bubble formation on the cell surface: membraneblebbing) are also observed. Also, A phenomenon in which DNA is fragmented due to the nuclease activity is also observed, and the cells themselves form cell fragments called apoptotic bodies, and the formed apoptotic bodies are rapidly mobilized by surrounding cells and macrophages. It is phagocytosed and apoptosis occurs. Therefore, apoptosis can be confirmed by, for example, fragmentation of DNA extracted from cells and morphological observation of the cells.
本発明の薬剤を医薬として使用する場合には、 一般的には、 有効成分としての ΙΤΠαと製剤用添加物 （担体、 賦形剤など） とを含む医薬組成物の形態で提供さ れる。  When the drug of the present invention is used as a medicine, it is generally provided in the form of a pharmaceutical composition containing Δα as an active ingredient and a pharmaceutical additive (carrier, excipient, etc.).
本発明の薬剤は、 ヒ トを含む哺乳動物に医薬として投与することができる。 本 発明の薬剤の投与経路は特に限定されず、 経口投与または非経口投与 （例えば、 筋肉内投与、 静脈内投与、 皮下投与、 腹腔内投与、 鼻腔などへの粘膜投与、 また は吸入投与など） の何れでもよい。  The agent of the present invention can be administered as a medicament to mammals including humans. The administration route of the drug of the present invention is not particularly limited, and is orally or parenterally administered (for example, intramuscular administration, intravenous administration, subcutaneous administration, intraperitoneal administration, mucosal administration to nasal cavity, or inhalation administration). Any of
本発明の薬剤の形態は特に限定されず、経口投与のための製剤としては例えば、 錠剤、 カプセル剤、 細粒剤、粉末剤、顆粒剤、液剤、 シロップ剤などが挙げられ、 非経口投与のための製剤としては例えば、 注射剤、 点滴剤、 座剤、 吸入剤、 経粘 膜吸収剤、 経皮吸収剤、 点鼻剤、 点耳剤などが挙げられる。  The form of the drug of the present invention is not particularly limited, and examples of preparations for oral administration include tablets, capsules, fine granules, powders, granules, liquids, syrups, and the like. Preparations include, for example, injections, drops, suppositories, inhalants, transmucosal absorbers, transdermal absorbers, nasal drops, ear drops, and the like.
本発明の薬剤の形態、 使用すべき製剤用添加物、 製剤の製造方法などは、 いず れも当業者が適宜選択可能である。  Those skilled in the art can appropriately select the form of the drug of the present invention, the additive for the preparation to be used, the method for producing the preparation, and the like.
本発明の薬剤の投与量は、 患者の性別、 年齢または体重、 症状の重症度、 予防 または治療といった投与目的、 あるいは他の合併症状の有無などを総合的に考慮 して適宜選択することができる。 投与量は、 一般的には、 0. O O l gZk g 体重 日〜 l O O O/ gZk g体重/ ^日、 好ましくは 0. 001 /z g/k g体重 /日〜 100/i g k g体重/日である。  The dose of the drug of the present invention can be appropriately selected in consideration of the gender, age or weight of the patient, the severity of the symptoms, the administration purpose such as prevention or treatment, and the presence or absence of other complications. . The dosage is generally between 0.000 g / kg body weight / day and 0.1000 g / kg body weight / day, preferably between 0.001 / z g / kg body weight / day and 100 g / kg body weight / day.
本発明の薬剤はまた、 医薬としてだけではなく、 ΤΟΡΙΙα阻害剤又はアポトー シス誘導剤として実験用の試薬などとしても有用である。 本発明の薬剤を試薬と して使用する場合、 一般的には、 有効成分としての ΓΠΙαは適当な溶媒などに溶 解した形態で提供される。 ( 2 ) アンチセンスオリゴヌクレオチド The agent of the present invention is also useful not only as a pharmaceutical, but also as a 実 験 α inhibitor or an apoptosis inducer as an experimental reagent or the like. When the agent of the present invention is used as a reagent, ΓΠΙα as an active ingredient is generally provided in a form dissolved in an appropriate solvent or the like. (2) Antisense oligonucleotide
本発明は、 下記の何れかの塩基配列中の連続する 5から 1 0 0の塩基配列のァ ンチセンス配列から成るアンチセンスオリゴヌクレオチドに関する。  The present invention relates to an antisense oligonucleotide comprising an antisense sequence of 5 to 100 consecutive nucleotide sequences in any of the following nucleotide sequences.
( a ) 配列番号 2に記載の塩基配列列；又は  (a) the nucleotide sequence set forth in SEQ ID NO: 2; or
( b ) 配列番号 2に記載の塩基配列において 1から数個の塩基が欠失、 置換及び 又は挿入した塩基配列を有し、 TOPII c と相互作用してその活性を阻害するこ とができるタンパク質をコードする塩基配列：  (b) a protein having a nucleotide sequence in which one to several nucleotides are deleted, substituted and / or inserted in the nucleotide sequence of SEQ ID NO: 2, and which can interact with TOPIIc to inhibit its activity Nucleotide sequence encoding:
本明細書において 「配列番号 2に記載の塩基配列において 1から数個の塩基が 欠失、 置換及び 又は挿入した塩基配列」 における 「1から数個」 の範囲は特に は限定されないが、 例えば、 1から 6 0個、 好ましくは 1から 3 0個、 より好ま しくは 1から 2 0個、 さらに好ましくは 1から 1 0個、 特に好ましくは 1から 5 個程度を意味する。  In the present specification, the range of `` 1 to several '' in the `` base sequence in which one to several bases are deleted, substituted and / or inserted in the base sequence described in SEQ ID NO: 2 '' is not particularly limited, for example, It means about 1 to 60, preferably about 1 to 30, more preferably about 1 to 20, more preferably about 1 to 10, and particularly preferably about 1 to 5.
本発明のアンチセンスオリゴヌクレオチドは、 上記の何れかのアミノ酸配列を コードする D N A配列中の連続する 5から 1 0 0の塩基配列に対して相補的な、 またはハイブリダイズするヌクレオチドであって、 D NA又はR N Aのぃずれで あっても良く、 また機能に支障がない限りにおいて修飾されたものであっても良 レ、。  The antisense oligonucleotide of the present invention is a nucleotide that is complementary or hybridizes to a continuous 5 to 100 nucleotide sequence in a DNA sequence encoding any of the above amino acid sequences, It may be a difference between NA and RNA, and may be modified as long as the function is not hindered.
本明細書で言う 「アンチセンスオリゴヌクレオチド」 とは、 D N A又はm R N Aの所定の領域を構成するヌクレオチドに対応するヌクレオチドがすべて相補的 であるもののみならず、 D N A又は m R N Aとオリゴヌクレオチドとが安定にハ イブリダィズできる限り、 多少のミスマッチが存在してもよレ、。  As used herein, the term "antisense oligonucleotide" refers to not only those in which all nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are complementary. As long as stable hybridization is possible, there may be some mismatches.
本発明のアンチセンスオリゴヌクレオチドとしては、 例えば、 配列表の配列番 号 9に示す塩基配列 （TAGCAGGTCCGACAT) を有するァンチセンスオリゴヌクレオ チドが挙げられる。 本明細書での実施例では、 このような塩基配列のアンチセン スオリゴヌクレオチドは、非常に効果的に ΙΤΠ αの発現を抑制することができた。 なお、 本発明で用いるアンチセンスオリゴヌクレオチドは、 ΙΤΠ αの発現を抑制 できるものであれば、 上述したものに限定されない。 Examples of the antisense oligonucleotide of the present invention include an antisense oligonucleotide having a base sequence (TAGCAGGTCCGACAT) shown in SEQ ID NO: 9 in the sequence listing. In the examples in the present specification, the antisense oligonucleotide having such a base sequence was able to suppress the expression of ΙΤΠα very effectively. The antisense oligonucleotide used in the present invention suppresses the expression of ΙΤΠα. It is not limited to the above as long as it is possible.
なお、 アンチセンスオリゴヌクレオチドは、 修飾されていてもよい。 適当な修 飾を施すことにより、 当該アンチセンスオリゴヌクレオチドは生体内で分解され にくくなり、 より安定して ΙΤΠ αを阻害できるようになる。 このような修飾され たオリゴヌクレオチドとしては、 S—オリゴ型 （ホスフォロチォェ一ト型）、 C - 5チアゾール型、 D—オリゴ型 （フォスフォジエステル型）、 Μ—オリゴ型 （メチ ルフォスフォネィト型）、ペプチド核酸型、 リン酸ジエステル結合型、 C一 5プロ ピニルピリミジン型、 2—Ο—プロピルリボース、 2 ' —メ トキシェトキシリボ ース型等の修飾型のアンチセンスオリゴヌクレオチドが挙げられる。  Note that the antisense oligonucleotide may be modified. By performing an appropriate modification, the antisense oligonucleotide is less likely to be degraded in vivo, and ΙΤΠα can be more stably inhibited. Such modified oligonucleotides include S-oligo type (phosphorothioate type), C-5 thiazole type, D-oligo type (phosphodiester type), and フ ォ -oligo type (methylphosphonate type). ), Peptide nucleic acid type, phosphodiester bond type, C-15 propynylpyrimidine type, 2-Ο-propylribose, 2'-methoxetoxyribose type and other modified antisense oligonucleotides. Can be
さらに、 アンチセンスオリゴヌクレオチドとしては、 リン酸基を構成する酸素 原子の少なくとも一部がィォゥ原子に置換、 修飾されているものでもよレ、。 この ようなアンチセンスオリゴヌクレオチドは、 ヌクレアーゼ耐性、 水溶性、 R N A への親和性に特に優れている。 リン酸基を構成する酸素原子の少なくとも一部が ィォゥ原子に置換、 修飾されたアンチセンスオリゴヌクレオチドとしては、 例え ば、 S _オリゴ型等のオリゴヌクレオチドが挙げられる。  Furthermore, the antisense oligonucleotide may be one in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a zeo atom. Such antisense oligonucleotides are particularly excellent in nuclease resistance, water solubility, and affinity for RNA. Examples of the antisense oligonucleotide in which at least a part of the oxygen atom constituting the phosphate group is substituted and modified with an atom include, for example, S_oligo-type oligonucleotides.
また、 アンチセンスオリゴヌクレオチドの塩基数は、 5 0以下であることが好 ましく、 2 5以下であることがより好ましい。 塩基数があまりに多くなると、 ォ リゴヌクレオチドの合成の手間とコス トが増大し、 また、 収率も低下する。 さら に、 アンチセンスオリゴヌクレオチドの塩基数は 5以上であり、 9以上であるこ とが好ましい。 塩基数が 4以下の場合には、 標的遺伝子に対する特異性が低下し て好ましくないためである。  The number of bases of the antisense oligonucleotide is preferably 50 or less, more preferably 25 or less. If the number of bases is too large, the labor and cost of synthesis of the oligonucleotide increase, and the yield also decreases. Further, the number of bases of the antisense oligonucleotide is 5 or more, and preferably 9 or more. When the number of bases is 4 or less, the specificity for the target gene is decreased, which is not preferable.
本発明のアンチセンスオリゴヌクレオチド （又はその誘導体） は常法によって 合成することができ、 例えば、 市販の D N A合成装置 （例えば A p p 1 i e d B i o s y s t e m s社製など） によって容易に合成することができる。 合成法は ホスホロアミダイトを用いた固相合成法、 ハイ ドロジェンホスホネートを用いた 固相合成法などで得ることができる。  The antisense oligonucleotide (or derivative thereof) of the present invention can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available DNA synthesizer (for example, Applied Biostems). The synthesis method can be obtained by a solid phase synthesis method using a phosphoramidite, a solid phase synthesis method using a hydrogen phosphonate, or the like.
本発明のアンチセンスオリゴヌクレオチドは、 ΙΤΠ αの阻害剤、 ΤΟΡΠ αの発 現増強剤、 アポトーシス誘導剤、 並びに抗癌剤として使用することができる。 な お、 本発明のアンチセンスオリゴヌクレオチドは、 癌細胞に特異的にアポトーシ スを誘導することにより抗癌作用を発揮することができるものと考えられる。 本発明のアンチセンスオリゴヌクレオチドを医薬として使用する場合には、 一 般的には、 アンチセンスオリゴヌクレオチドと製剤用添加物 （担体、賦形剤など） とを含む医薬組成物の形態で提供される。 The antisense oligonucleotide of the present invention comprises an inhibitor of ΙΤΠα, It can be used as a current enhancer, an apoptosis inducer, and an anticancer agent. It is considered that the antisense oligonucleotide of the present invention can exert an anticancer action by specifically inducing apoptosis in cancer cells. When the antisense oligonucleotide of the present invention is used as a medicament, it is generally provided in the form of a pharmaceutical composition containing the antisense oligonucleotide and a pharmaceutical additive (carrier, excipient, etc.). You.
本発明のアンチセンスオリゴヌクレオチドは、 ヒ トを含む哺乳動物に医薬とし て投与することができる。 本発明のアンチセンスオリゴヌクレオチドの投与経路 は特に限定されず、 経口投与または非経口投与 （例えば、 筋肉内投与、 静脈内投 与、 皮下投与、 腹腔内投与、 鼻腔などへの粘膜投与、 または吸入投与など） の何 れでもよレ、。  The antisense oligonucleotide of the present invention can be administered as a medicine to mammals including humans. The administration route of the antisense oligonucleotide of the present invention is not particularly limited. For example, oral administration or parenteral administration (for example, intramuscular administration, intravenous administration, subcutaneous administration, intraperitoneal administration, mucosal administration to nasal cavity, or inhalation) Administration, etc.).
アンチセンスオリゴヌクレオチドの製剤形態は特に限定されず、 経口投与のた めの製剤としては例えば、 錠剤、 カプセル剤、 細粒剤、 粉末剤、 顆粒剤、 液剤、 シロップ剤などが挙げられ、 非経口投与のための製剤としては例えば、 注射剤、 点滴剤、 座剤、 吸入剤、 経粘膜吸収剤、 経皮吸収剤、 点鼻剤、 点耳剤などが挙げ られる。 アンチセンスオリゴヌクレオチドを含む薬剤の形態、 使用すべき製剤用 添加物、 製剤の製造方法などは、 いずれも当業者が適宜選択可能である。 さらに 持続性、膜透過性を高めるアンチセンス封入素材を用いることもできる。例えば、 リボゾーム、 ポリ一 L—リジン、 リピッド、 コレステロール、 リボフヱクチル又 はこれらの誘導体が挙げられる。  The formulation of the antisense oligonucleotide is not particularly limited, and examples of formulations for oral administration include tablets, capsules, fine granules, powders, granules, solutions, syrups, and the like. Formulations for administration include, for example, injections, drops, suppositories, inhalants, transmucosal absorbents, transdermal absorbents, nasal drops, ear drops and the like. Those skilled in the art can appropriately select the form of the drug containing the antisense oligonucleotide, the additive for the drug to be used, the method for producing the drug, and the like. Further, an antisense encapsulating material that enhances durability and membrane permeability can be used. For example, ribosome, poly-L-lysine, lipid, cholesterol, ribopectyl or derivatives thereof can be mentioned.
アンチセンスオリゴヌクレオチドの投与量は、 患者の性別、 年齢または体重、 症状の重症度、 予防または治療といった投与目的、 あるいは他の合併症状の有無 などを総合的に考慮して適宜選択することができる。投与量は、一般的には、 0 . 1 μ g / k g体重/日〜 l O O m g Z k g体重/日、 好ましくは 0 · 1 μ g Z k g体重 Z日〜 1 O m g Z k g体重/日である。  The dosage of the antisense oligonucleotide can be appropriately selected in consideration of the patient's sex, age or weight, the severity of the symptoms, the administration purpose such as prevention or treatment, and the presence or absence of other complications. . The dose is generally 0.1 μg / kg body weight / day to 100 mg Z kg body weight / day, preferably 0.1 μg Z kg body weight Z day to 1 O mg Z kg body weight / day. It is.
( 3 ) ΙΤΙΙ αを認識する抗体 本発明の抗体は、 以下の何れかのアミノ酸配列を有する ΙΤΠ αを認識する。 ( a ) 配列番号 1に記載のァミノ酸配列；又は (3) Antibodies that recognize ΙΤΙΙα The antibody of the present invention recognizes ΙΤΠα having any of the following amino acid sequences. (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のァミノ酸配列において 1から数個のァミノ酸が欠失、 置換及び/又は挿入したァミノ酸配列を有し、 ΤΟΡΠ αと相互作用してその活性 を阻害することができるァミノ酸配列：  (b) One or several amino acids in the amino acid sequence described in SEQ ID NO: 1 have a deleted, substituted and / or inserted amino acid sequence, and interact with ΤΟΡΠα to inhibit its activity. Amino acid sequences that can:
本発明の抗体はポリクローナル抗体でもモノクローナル抗体でもよく、 その作 製は定法により行なうことができる。  The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and its production can be performed by a conventional method.
例えば、 ΙΤΠ αを認識するポリクローナル抗体は、 ΙΤΠ α又はその部分ぺプチ ドを抗原として哺乳動物を免疫感作し、 該哺乳動物から血液を採取し、 採取した 血液から抗体を分離 '精製することにより得ることができる。 例えば、 マウス、 ハムスター、 モルモッ ト、 ニヮトリ、 ラット、 ゥサギ、 ィヌ、 ャギ、 ヒッジ、 ゥ シ等の哺乳動物を免疫することができる。免疫感作の方法は当業者に公知であり、 例えば抗原を 1回以上投与することにより行うことができる。 抗原投与は、 例え ば 7〜3 0 S間隔で 2〜3回投与すればよレ、。 投与量は 1回につき、 例えば抗原 約 0 . 0 5〜2 m g程度とすることができる。 投与経路も特に限定されず、 皮下 投与、 皮内投与、 腹膜腔内投与、 静脈内投与、 筋肉内投与等を適宜選択すること ができるが、 静脈内、 腹膜腔内もしくは皮下に注射することにより投与すること が好ましい。 また、 抗原は適当な緩衝液、 例えば完全フロイントアジュバントま たは水酸化アルミニウム等の通常用いられるアジュバントを含有する適当な緩衝 液に溶解して用いることができるが、 投与経路や条件等に応じてアジュバントを 使用しない場合もある。  For example, a polyclonal antibody that recognizes ΙΤΠα is obtained by immunizing a mammal with ΙΤΠα or a partial peptide thereof as an antigen, collecting blood from the mammal, and separating and purifying the antibody from the collected blood. Can be obtained by For example, mammals such as mice, hamsters, guinea pigs, chickens, rats, egrets, dogs, goats, sheep, and birds can be immunized. Methods for immunization are known to those skilled in the art, and can be performed, for example, by administering the antigen one or more times. The antigen may be administered, for example, two to three times at an interval of 7 to 30 S. The dose may be, for example, about 0.05 to 2 mg per antigen. The route of administration is not particularly limited, and can be selected as appropriate from subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc., by injecting intravenously, intraperitoneally or subcutaneously. Preferably, it is administered. The antigen can be used by dissolving it in an appropriate buffer, for example, an appropriate buffer containing a commonly used adjuvant such as complete Freund's adjuvant or aluminum hydroxide, depending on the administration route and conditions. In some cases, no adjuvant is used.
免疫感作した哺乳動物を一定期間飼育した後、 該哺乳動物の血清をサンプリン グし、 抗体価を測定する。 抗体価が上昇してきたら、 例ぇば1 0 0 μ g〜1 0 0 0 μ gの抗原を用いて追加免疫を行なう。 最後の投与から 1〜2ヶ月後に免疫感 作した哺乳動物から血液を採取して、 該血液を、 例えば遠心分離、 硫酸アンモニ ゥムまたはポリエチレンダリコールを用いた沈澱、ゲルろ過クロマトグラフィー、 イオン交換クロマトグラフィー、 ァフィ二テイクロマトグラフィ一等のクロマト グラフィ一等の常法によって分離.精製することにより、 ポリクロ一ナル抗血清 として、本発明のタンパク質を認識するポリクローナル抗体を得ることができる。 After the immunized mammal is bred for a certain period, the serum of the mammal is sampled, and the antibody titer is measured. When the antibody titer increases, booster immunization is performed using, for example, 100 μg to 100 μg of the antigen. One to two months after the last administration, blood is collected from the immunized mammal, and the blood is collected, for example, by centrifugation, precipitation using ammonium sulfate or polyethylene daricol, gel filtration chromatography, ion exchange. Chromatography such as chromatography and affinity chromatography A polyclonal antibody recognizing the protein of the present invention can be obtained as a polyclonal antiserum by separating and purifying by a conventional method such as graphy.
ΙΤΙΙαを認識するモノクローナル抗体のグロブリンタイプは特に限定されず、 例えば I gG、 I gM、 I gA、 I g E、 I g D等が挙げられる。 モノクローナ ル抗体を産生する細胞株は特に制限されないが、 例えば、 抗体産生細胞とミエ口 一マ細胞株との細胞融合によりハイプリ ドーマとして得ることができる。 本発明 のモノクローナル抗体を産生するハイプリ ドーマは、 以下のような細胞融合法に よって得ることができる。  The globulin type of the monoclonal antibody that recognizes ΙΤΙΙα is not particularly limited, and examples include IgG, IgM, IgA, IgE, and IgD. The cell line that produces the monoclonal antibody is not particularly limited. For example, it can be obtained as a hybridoma by cell fusion between the antibody-producing cell and the Myeoma cell line. The hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.
抗体産生細胞としては、 免疫された動物からの脾細胞、 リンパ節細胞、 Bリン パ球等を使用する。 抗原としては、 本発明のタンパク質又はその部分ペプチドを 使用する。 免疫動物としてはマウス、 ラット等を使用でき、 これらの動物への抗 原の投与は常法により行う。 例えば完全フロインドアジュバント、 不完全フロイ ンドアジュバントなどのアジュバントと抗原である本発明のタンパク質との懸濁 液もしくは乳化液を動物の静脈、 皮下、 皮内、 腹腔内等に数回投与することによ つて動物を免疫化する。 免疫化した動物から抗体産生細胞として例えば脾細胞を 取得し、 これとミエ口一マ細胞とを公知の方法 （G.Kohler et al . , Nature, 256 495(1975)) により融合してハイプリ ドーマを作製することができる。  As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used. As the antigen, the protein of the present invention or its partial peptide is used. Mice, rats and the like can be used as immunized animals, and administration of the antigen to these animals is performed by a conventional method. For example, a suspension or emulsion of an adjuvant, such as complete Freund's adjuvant or incomplete Freund's adjuvant, and the protein of the present invention, which is an antigen, is administered several times to the vein, subcutaneous, intradermal, intraperitoneal, etc. of an animal. To immunize animals. For example, spleen cells are obtained as antibody-producing cells from the immunized animal, and the spleen cells are fused with myeloid cells by a known method (G. Kohler et al., Nature, 256 495 (1975)) to obtain a hybridoma. Can be produced.
細胞融合に使用するミエローマ細胞株としては、 例えばマウスでは P 3X63 Ag 8、 P 3U1株、 S p 2/0株などが挙げられる。 細胞融合を行なうに際し ては、 ポリエチレングリコール、 センダイウィルスなどの融合促進剤を用い、 細 胞融合後のハイブリ ドーマの選択にはヒポキサンチン ·アミノプテリン ·チミジ ン （HAT) 培地を常法に従って使用する。 細胞融合により得られるハイプリ ド 一マは限界希釈法等によりクローユングする。 さらに必要に応じて、 ΙΤΠο;又は その部分べプチドを用いた酵素免疫測定法によりスクリ一二ングを行なうことに より、 ΙΤΠαを特異的に認識するモノクローナル抗体を産生する細胞株を得るこ とができる。  Examples of myeloma cell lines used for cell fusion include the P3X63 Ag8, P3U1 and Sp2 / 0 strains in mice. Cell fusion is performed using a fusion promoter such as polyethylene glycol or Sendai virus.Hypoxanthine / aminopterin / thymidine (HAT) medium is used for selection of hybridomas after cell fusion in a conventional manner. . Hybridomas obtained by cell fusion are cloned by a limiting dilution method or the like. Further, if necessary, screening by enzyme immunoassay using た ο; or a partial peptide thereof can obtain a cell line that produces a monoclonal antibody that specifically recognizes ΙΤΠα. it can.
このようにして得られたハイブリ ドーマから目的と'するモノクローナル抗体を 製造するには、通常の細胞培養法や腹水形成法により該ハイプリ ドーマを培養し、 培養上清あるいは腹水から該モノクローナル抗体を精製すればよい。 培養上清も しくは腹水からのモノクローナル抗体の精製は、常法により行なうことができる。 例えば、 硫安分画、 ゲルろ過、 イオン交換クロマトグラフィー、 ァフィ二ティー クロマトグラフィーなどを適宜組み合わせて使用できる。 From the hybridoma thus obtained, a monoclonal antibody of interest is For production, the hybridoma may be cultured by a usual cell culture method or ascites formation method, and the monoclonal antibody may be purified from the culture supernatant or ascites. Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used in an appropriate combination.
本発明の抗体を用いて、 ΙΤΠ αを免疫測定するための方法としては、 例えば酵 素免疫測定法、 ラジオィムノアツセィ、 蛍光免疫測定法、 発光免疫測定法等を挙 げることができる。  Examples of the method for immunoassay of ΙΤΠα using the antibody of the present invention include enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, and luminescence immunoassay. .
また、 上記した抗体の断片も本発明の範囲内である。 抗体の断片としては、 F ( a b ' ) 2フラグメント、 F a b， フラグメント等が挙げられる。  Also, fragments of the above antibodies are within the scope of the present invention. Antibody fragments include F (ab ') 2 fragments, Fab, fragments and the like.
さらに、 上記した抗体の標識抗体も本発明の範囲内である。 即ち、 上記のよう にして作製した本発明の抗体は標識して使用することができる。 抗体の標識の種 類及び標識方法は当業者に公知である。 例えば、 西洋ヮサビペルォキシダーゼ又 はアルカリホスファターゼなどの酵素標識、 F I T C (フルォレセインイソチォ シァネート） 又は T R I T C (テトラメチルローダミン Bイソチオシァネート） 等の蛍光標識、 コロイ ド金属および着色ラテックスなどの呈色物質による標識、 ピオチンなどのアブイ二ティー標識、 あるいは1 2 5 Iなどの同位体標識などを挙 げることができる。 本発明の標識抗体を用いた酵素抗体法、 免疫組織染色法、 免 疫ブロット法、 直接蛍光抗体法又は間接蛍光抗体法等の分析は当業者に周知の方 法により行うことができる。 Furthermore, labeled antibodies of the above-mentioned antibodies are also within the scope of the present invention. That is, the antibody of the present invention prepared as described above can be labeled and used. The types and methods of labeling antibodies are known to those skilled in the art. For example, enzyme labels such as horseradish peroxidase or alkaline phosphatase, fluorescent labels such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocynate), colloid metal and colored latex labeling with color substances such as roasting two tea labels such Piochin, or 1 2 5 I and isotopic labels, such as can ani gel. Analysis using the labeled antibody of the present invention, such as an enzyme antibody method, an immunohistochemical staining method, an immunoblotting method, a direct fluorescent antibody method or an indirect fluorescent antibody method, can be performed by a method well known to those skilled in the art.
( 4 ) ΙΤΠ αの阻害剤のスクリーニング方法 (4) Screening method for ΙΤΠα inhibitor
本発明は、 被験物質の存在下において、 下記の何れかのアミノ酸配列を有する ΙΤΠ αと、 ΤΟΡΠ αとの相互作用を測定し、 該相互作用を阻害する物質を選択す ることを特徴とする、 ΙΤΠ αの阻害剤のスクリーニング方法に関する。  The present invention is characterized in that in the presence of a test substance, an interaction between ΙΤΠα having any of the following amino acid sequences and ΤΟΡΠα is measured, and a substance that inhibits the interaction is selected. , に 関 す る α screening method.
( a ) 配列番号 1に記載のァミノ酸配列；又は  (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のァミノ酸配列において 1から数個のァミノ酸が欠失、 置換及び/又は挿入したアミノ酸配列を有し、 ΤΟΡΠ αと相互作用してその活性 を阻害することができるアミノ酸配列： (b) 1 to several amino acids are deleted in the amino acid sequence described in SEQ ID NO: 1, An amino acid sequence having a substituted and / or inserted amino acid sequence and capable of interacting with ΤΟΡΠα to inhibit its activity:
ΙΤΙΙ αと、 ΤΟΡΠ αとの相互作用を測定し、 該相互作用を阻害する物質を選択す るためには、 例えば、 ΙΤΠ α; の活性 （ΤΟΡΠ α活性の阻害作用） を阻害する物質 を選択することができる。 具体的なスクリーニング系としては、 例えば、 基質 D Ν Α、 ΤΟΡΙΙ α , ΙΤΙΙ α , さらに被験物質を加えて、 ΤΟΡΠ αの活性が回復するか どうかを検出するスクリーニング系が挙げられる。  In order to measure the interaction between ΙΤΙΙα and ΤΟΡΠα and to select a substance that inhibits the interaction, for example, select a substance that inhibits the activity of ΙΤΠα; can do. As a specific screening system, for example, a screening system for detecting whether or not the activity of ΤΟΡΠα is restored by adding a substrate D Ν, ΤΟΡΙΙα, α and a test substance can be mentioned.
本発明で用いる被験物質としては任意の物質を使用することができ、 その種類 は特に限定されない。 被験物質の具体例としては、 低分子化合物、 抗体又はオリ ゴヌクレオチドでもよいし、 天然物抽出物でもよく、 あるいは化合物ライブラリ 一、 ファージディスプレーライブラリーもしくはコンビナトリアルライブラリー でもよい。 化合物ライブラリ一の構築は当業者に公知であり、 また市販の化合物 ライブラリ一を使用することもできる。被験物質としては、低分子化合物、抗体、 オリゴヌクレオチド、 又はそれらのライブラリーが好ましい。  As the test substance used in the present invention, any substance can be used, and its type is not particularly limited. Specific examples of the test substance may be a low molecular weight compound, an antibody or an oligonucleotide, a natural product extract, or a compound library, a phage display library or a combinatorial library. Construction of compound libraries is known to those skilled in the art, and commercially available compound libraries can also be used. The test substance is preferably a low molecular compound, an antibody, an oligonucleotide, or a library thereof.
また、 本発明のスクリーニング方法により得られる、 ΙΤΠ αの阻害剤も本発明 の範囲内である。  Further, an inhibitor of ΙΤΠα obtained by the screening method of the present invention is also within the scope of the present invention.
本発明のスクリーニング方法では、 被験物質の存在下において、 ΙΤΙΙ αと Τ0ΡΠ αとの相互作用を測定する。 相互作用の測定方法は特に限定されない。 また、 上 記相互作用それ自体を直接的に測定してもよいし、 ΤΟΡΠ α活性を測定すること により、 上記相互作用を間接的に測定することもできる。  In the screening method of the present invention, the interaction between ΙΤΙΙα and Τ0ΡΠα is measured in the presence of a test substance. The method for measuring the interaction is not particularly limited. Further, the above-mentioned interaction itself may be directly measured, or the above-mentioned interaction may be indirectly measured by measuring ΤΟΡΠα activity.
例えば、 ΤΟΡΠ α活性の測定方法としては、 例えば、 ΤΟΡΠ αの Relaxation活性 及び Decatenation活性に対する阻害作用を測定することにより評価することが でき、 その測定方法は前述の通りである。  For example, as a method for measuring ΤΟΡΠα activity, it can be evaluated by, for example, measuring the inhibitory effect of ΤΟΡΠα on Relaxation activity and Decatenation activity, and the measuring method is as described above.
一般的には、 同一のアツセィ系を被験物質の非存在下でも行い、 被験物質の存 在下の場合と非存在下の場合の両者における上記相互作用を測定し、 両者を比較 することにより、 被験物質が上記相互作用を阻害しているかどうかを判別するこ とが好ましい。 以下の実施例により本発明をさらに具体的に説明するが、 本発明は実施例によ つて限定されることはない。 実施例 In general, the same Atsushi system is performed in the absence of the test substance, and the above-mentioned interaction in the presence and absence of the test substance is measured. It is preferable to determine whether a substance is inhibiting the above interaction. The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. Example
実施例 1 Example 1
( I ) 実験方法  (I) Experimental method
( i ) 使用した細胞株リスト  (i) List of cell lines used
Sf9, High Five (両者は昆虫細胞)  Sf9, High Five (both are insect cells)
HeLa, COS- 1, HL60 (東大医科研より分与）、 Jurcat (東大医科研より分与） また、 以下の 4株は理研ジーンバンク ·細胞開発銀行より購入した： CW- 2 (大 腸癌細胞株） ， TUHR4TKB (腎癌細胞株) , TGBC2TKB (胆嚢癌細胞株) , TT1TKB (直 腸癌細胞株）  HeLa, COS- 1, HL60 (distributed from the University of Tokyo Medical Research Institute), Jurcat (distributed from the University of Tokyo Medical Research Institute) The following four strains were purchased from RIKEN Genebank and Cell Development Bank: CW-2 (colorectal cancer cell line) ), TUHR4TKB (kidney cancer cell line), TGBC2TKB (gallbladder cancer cell line), TT1TKB (rectal cancer cell line)
(i i) 遺伝子のクローニング  (ii) Gene cloning
HeLa細胞由来 cDNAライブラリーを铸型にして、 以下のプライマーを用いて PCR (94°Cで 30秒後、 94°Cで 30秒、 50°Cで 1分及ぴ 72°Cで 2. 5分を 30サイクル行 レ、、 さらに 72でで 3分反応） を行った。  The HeLa cell-derived cDNA library was converted to type II and PCR was performed using the following primers (30 seconds at 94 ° C, 30 seconds at 94 ° C, 1 minute at 50 ° C, and 2.5 minutes at 72 ° C). The reaction was carried out for 30 minutes, followed by 72 minutes for 3 minutes.
センス ； 5' -TTGGTACCATGTCGGACCTGCTACTACTGGGCCTGATT-3' (配列番号 3 )、 ァンチセンス ； 5' -TTGGTACCTTACTCCTTGCCCTTCTCAGGGGCAGT-3' (配列番号 4 ) 増幅させた DNAフラグメントをプラスミ ドベクター SRHisBの Kpnl部位に組み 込み、 ΙΤΠ α発現プラスミ ド （pRCl) を構築した。 Sense; 5'-TTGGTACCATGTCGGACCTGCTACTACTGGGCCTGATT-3 '(SEQ ID NO: 3), Antisense; 5'-TTGGTACCTTACTCCTTGCCCTTCTCAGGGGCAGT-3' (SEQ ID NO: 4) (PRCl).
(ii i) ΙΤΠ αの細胞内への導入  (ii i) Introduction of ΙΤΠα into cells
ΙΤΠ αは、 COS- 1細胞 (African green monkey kidney cell line CV- 1細胞を origin- defective SV40 DNAで転換した細胞） 内で発現させた。 500 μ 1 の COS - 1 細胞 （6 X 106細胞/ ml) に 16 w gの pRClを加えて 10分間氷中に静置後、 エレク トロポレーシヨン （276 V、 975 F) を行った。 37°Cで 10分間 （5% C02 ) 保温後、 9 mlの培地 （D_MEM、 10%FBS) に添力！]し、 450 μ 1 を Chamber slides (Nunc) に 分注して、 3日後後、 間接蛍光抗体法により細胞内の局在を測定した。 (iv) 間接蛍光抗体法 ΙΤΠα was expressed in COS-1 cells (African green monkey kidney cell line CV-1 cells transformed with origin-defective SV40 DNA). 16 μg of pRCl was added to 500 μl of COS-1 cells (6 × 10 6 cells / ml), allowed to stand on ice for 10 minutes, and electroporated (276 V, 975 F). 37 ° C for 10 min (5% C0 2) After incubation,添力to 9 ml of medium (D_MEM, 10% FBS)! Then, 450 μl was dispensed into Chamber slides (Nunc), and three days later, intracellular localization was measured by the indirect fluorescent antibody method. (iv) Indirect fluorescent antibody method
細胞を phosphate- buffer saline (PBS)で洗浄後、 3. 7%ホルムアルデヒ ド （PBS 中） を加えて 10 分間氷上に静置して細胞を固定した。 その後、 PBS で洗浄して 50、 75、 95%エタノールを順次添カ卩して最後に PBS で洗浄した。 これまでの操作 は、全て氷上にて行った。 以後の操作は室温にて行った。細胞に Blocking buffer (5% 正常ャギ血清 （PBS 中） ） を添加して 30 分間静置した。 その後、 1 次抗体 After washing the cells with phosphate-buffer saline (PBS), 3.7% formaldehyde (in PBS) was added and the cells were fixed on ice for 10 minutes to fix the cells. After that, the cells were washed with PBS, added with 50, 75, and 95% ethanol sequentially, and finally washed with PBS. All operations so far were performed on ice. Subsequent operations were performed at room temperature. Blocking buffer (5% normal goat serum (in PBS)) was added to the cells and allowed to stand for 30 minutes. Then the primary antibody
(anti-T7 tag (1： 3000に希釈）， anti— T0P2 a Ab— 2 (1. 6 μ g/400 μ 1； Neomarkers) antibody in blocking buffer) を加えて 1時間静置した。 そして PBSで 5分間 ずつ 3 回洗浄した。 次に、 FITC (1. 5 μ g/ml) または、 Texas red (1. 5 μ g/ral) -conjugated 2次抗体を加えて 1時間静置した。 PBSで 5分間ずつ 3回洗浄 後、 PBSに 1 μ gの bis - benzimide (Hoechst 33258)を加えた溶液を添加して DNA 染色を行った。 その後、 サンプルは Vectorshield (Vector Inc. ) で保存して、 01y即 us PROVIS AX70蛍光顕微鏡で観察した。 (anti-T7 tag (diluted 1: 3000), anti-T0P2 a Ab-2 (1.6 μg / 400 μ1; Neomarkers) antibody in blocking buffer) were added and left for 1 hour. Then, the cells were washed three times with PBS for 5 minutes each. Next, FITC (1.5 μg / ml) or Texas red (1.5 μg / ral) -conjugated secondary antibody was added and left for 1 hour. After washing with PBS three times for 5 minutes each, a solution obtained by adding 1 μg of bis-benzimide (Hoechst 33258) to PBS was added, and DNA staining was performed. Thereafter, the samples were stored in Vectorshield (Vector Inc.) and observed immediately on a 01y us PROVIS AX70 fluorescence microscope.
( v ) 抗体作製と精製  (v) Antibody production and purification
ITII cのアミノ酸配列 246- GASSRGWDDGDTRSEHSYSESG - 267 (ペプチド 1 ) (配列 番号 5 )、 302- LWEPTAPEKGKE- 313 (ぺプチド 2 ) (配列番号 6 ) を抗原としてゥサ ギを用いて抗血清を採取した。 各ペプチドを用いてペプチドカラムを調製し、 抗 血清から抗体を精製した。 4 ralの抗血清から、 ペプチド 1由来の抗体 1. 38 mg/ral (5 ml)とペプチド 2由来の抗体 2. 4 rag/ml (5 ml)を調製した。  Antiserum was collected from rabbits using the amino acid sequence of ITII c 246- GASSRGWDDGDTRSEHSYSESG-267 (peptide 1) (SEQ ID NO: 5) and 302-LWEPTAPEKGKE-313 (peptide 2) (SEQ ID NO: 6) as antigens. A peptide column was prepared using each peptide, and the antibody was purified from the antiserum. From 4 ral antisera, 1.38 mg / ral (5 ml) of the antibody derived from peptide 1 and 2.4 rag / ml (5 ml) of the antibody derived from peptide 2 were prepared.
(vi) 昆虫細胞による ΙΤΙΙ αの発現系の構築  (vi) Construction of ΙΤΙΙα expression system by insect cells
蛋白質発現系には、 BAC-T0-BAC Baculovirus Expression Systems (Gibco, BRL) を用いて行った。 hgyrl遺伝子は、 以下のプライマーを設計して PCR法 （94°Cで 2分後、 94°Cで 30秒及び 68°Cで 3分を 30サイクル行い、 さらに 68°Cで 3 分反 応） によって増幅した。 铸型には pRClを 10 ng使用した。  BAC-T0-BAC Baculovirus Expression Systems (Gibco, BRL) was used as the protein expression system. For the hgyrl gene, design the following primers and perform PCR (2 minutes at 94 ° C, 30 cycles at 94 ° C for 30 seconds and 68 ° C for 30 minutes, and a further 3 minutes reaction at 68 ° C) Amplified by Type 铸 used 10 ng of pRCl.
センス ； 5' -CGCGGTCCGAAACCATGTCGGACCTGCTACTACTG-3' (配列番号 7 )、 ァンチセンス ； 5' -ATCGGACCGCTCCTTGCCCTTCTCA-3' (配列番号 8 ) Sense; 5'-CGCGGTCCGAAACCATGTCGGACCTGCTACTACTG-3 '(SEQ ID NO: 7); Antisense; 5'-ATCGGACCGCTCCTTGCCCTTCTCA-3' (SEQ ID NO: 8)
目的遺伝子を pFASTBAC Htbベクターの Rsr IIサイ トに組み込んで、 トランス ファーベクター （pRC2) を構築した。 8. 5ng の pRC2を 100 1の DH10BAC コン ピテント細胞に導入して 37°Cで 24時間培養した。 コロニーを 2 mlの LB mediumInsert the gene of interest into the Rsr II site of the pFASTBAC Htb vector and A fur vector (pRC2) was constructed. 8. 5 ng of pRC2 was introduced into 100 1 DH10BAC competent cells and cultured at 37 ° C for 24 hours. Remove colonies with 2 ml of LB medium
(50 /i g/ml カナマイシン， i i g/ml ゲンタマイシン， lO ^ g/ml テトラサイタリ ン） に培養後、 組み換え bacmid DNA を調製し、 40 μ 1 の ΤΕ に溶解させた。 50 units/mlぺニシリンと 50 1ストレプトマイシンを含む 2mlの SF-900 II SFM に SF9細胞を 1 X 106細胞 （6- well plate)に調製した。 27°Cで 1時間培養した 後、 0, 5, 10 1の bacmid DNAを 100 1 の抗生物質を含まない SF- 900 II SF に溶解させた。 6 μ 1の Cell FECTIN Reagentを他の 100 μ 1の抗生物質を含まな い SF-900 II SFMに添加し、 室温で 30分間ィンキュベートした。 その後、 抗生 物質を含まない 800 μ 1の SF- 900 II SF を加えて、 同様の培地で洗浄した細胞 に滴下した。 27°Cで 5時間培養した後、 2ml の抗生物質入りの SF- 900 II SFMを 加えて、 27°Cで 72時間培養した。 上清液を回収し、 3000 rpmで 5分間遠心後、 再度、 上清液を回収して組み換えウィルス液とした。 1 X 106細胞の SF9細胞に組 み換えウィルス液 500 lを加えて、 27°Cで 1時間培養した後、 1. 5 mlの抗生物 質入りの SF- 900 II SFMを加えた。 27°Cで 4日間培養して、 上清を遠心後、 同様 の操作を行い、 組み換えウィルスの増幅を行い、 109 PFU/mlのウィルス液を調製 した。 その際、 残った細胞からタンパク質を抽出して目的遺伝子の発現をウェス タンブロッテイングで確認した。 タンパク質の大量発現は、 SF9 細胞から High Five細胞にかえて 109 PFU/ralのウィルス液を加えて行った。 細胞を回収後に、 SDS-PAGEで蛋白質の発現を確認した。 (50 / ig / ml kanamycin, iig / ml gentamicin, lO ^ g / ml tetracitalin), and recombinant bacmid DNA was prepared and dissolved in 40 µl of DNA. SF9 cells were prepared in 1 × 10 6 cells (6-well plate) in 2 ml of SF-900 II SFM containing 50 units / ml of penicillin and 50 1 streptomycin. After culturing at 27 ° C. for 1 hour, 0, 5, 101 bacmid DNA was dissolved in 100 1 of SF-900 II SF without antibiotics. 6 μl of Cell FECTIN Reagent was added to another 100 μl of SF-900 II SFM without antibiotics and incubated for 30 minutes at room temperature. Thereafter, 800 μl of SF-900 II SF without antibiotics was added and added dropwise to cells washed with the same medium. After culturing at 27 ° C for 5 hours, 2 ml of SF-900II SFM containing antibiotics was added, and culturing was performed at 27 ° C for 72 hours. The supernatant was collected, centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected again to obtain a recombinant virus solution. 500 l of the recombinant virus solution was added to 1 × 10 6 SF9 cells, cultured at 27 ° C. for 1 hour, and then 1.5 ml of SF-900II SFM containing antibiotics was added. After culturing at 27 ° C for 4 days, the supernatant was centrifuged, and the same operation was carried out to amplify the recombinant virus to prepare a virus solution of 109 PFU / ml. At that time, proteins were extracted from the remaining cells, and the expression of the target gene was confirmed by Western blotting. Overexpression of protein was performed by adding virus solution for 10 9 PFU / ral in place of High Five cells from SF9 cells. After collecting the cells, the expression of the protein was confirmed by SDS-PAGE.
(νϋ) ΙΤΠ αの精製  (νϋ) ΙΤΠ α Purification
ペプチド 2由来の抗体 12 mg を CNBr - activated Sepharose4B (膨潤 1 ml) に 結合させたぺプチド抗体を調製した。目的のタンパク質を発現している High Five 細胞 （1 X 108細胞） を回収後、 2 mlの RX buffer (100 raM KC1, 3 mM NaCl, 3. 5 m MgCl 2 , 1. 25 mM EGTA, 100 raM HEPES (pH 7. 3) , 1 mM PMSF)に縣濁して超音 波破砕した。 40, 000 rpmで 1時間遠心した後に、 RX bufferで平衡化した抗体力 ラムに添加して、 4°Cで 50分間静置後、 RX buffer, 0. 1% Triton X- 100で順次洗 浄した。 50 mM Glysine を含む 0.1% Triton X- 100、 5 mlで溶出してすぐに中和 した後、 50 mM Tris-HCl (pH 7.5)， 25% Glycerol, 0.5 mM DTT， 1 mM EDTA で 透析した。 A peptide antibody was prepared by binding 12 mg of the antibody derived from peptide 2 to CNBr-activated Sepharose 4B (1 ml of swelling). After collecting High Five cells (1 × 10 8 cells) expressing the target protein, 2 ml of RX buffer (100 raM KC1, 3 mM NaCl, 3.5 mM MgCl 2 , 1.25 mM EGTA, 100 The suspension was suspended in raM HEPES (pH 7.3, 1 mM PMSF) and sonicated. After centrifugation at 40,000 rpm for 1 hour, add to the antibody buffer equilibrated with RX buffer, and stand at 4 ° C for 50 minutes, then wash sequentially with RX buffer, 0.1% Triton X-100. Was cleaned. Immediately after elution with 5 ml of 0.1% Triton X-100 containing 50 mM Glysine for neutralization, the mixture was dialyzed against 50 mM Tris-HCl (pH 7.5), 25% Glycerol, 0.5 mM DTT, and 1 mM EDTA.
(viii) ΤΟΡΠαの活性測定  (viii) Activity measurement of 測定 α
Decatenation 活性の測定は、 基質にキネトプラスト DNA (0.175 μ g/assay) を用い、 Relaxation活性の測定は、 基質に pUC19 (0.3 μ g/assay) を用いた。 ともに、 ΤΟΡΙΙαを lU/assay (30μ 1; 50 mM Tris-HCl (pH 7.5)， 120 mM KC1, 10 mM MgCl2, 0.5 mM ATP, 0.5 mM DTT, 30 μ g/ml nuclease free BSA) 使用し、 37°Cで 60分間反応させた。 Decatenation activity was measured using kinetoplast DNA (0.175 μg / assay) as a substrate, and Relaxation activity was measured using pUC19 (0.3 μg / assay) as a substrate. In both cases, ΤΟΡΙΙα was used in lU / assay (30μ 1; 50 mM Tris-HCl (pH 7.5), 120 mM KC1, 10 mM MgCl 2 , 0.5 mM ATP, 0.5 mM DTT, 30 μg / ml nuclease free BSA) The reaction was performed at 37 ° C for 60 minutes.
(ix) プラズモン共鳴法を用いた相互作用の解析  (ix) Analysis of interaction using plasmon resonance method
プラズモン共鳴法による測定は、 IAsys plusを用いて行った。 固相化用のキュ ベットには CMデキス トランキュベットを使用して、 ΙΤΠαを固相化した。 固相 化法および測定方法は、 IAsys plus添付のプロ トコールに準じて行い、 267 arc seconds相当固相化した (200 arc seconds = 1 ng/mm2)0 TOPII c を 2· 9〜22· 2nM の範囲内で添加し、 このデータを FASTfit にて解析し、 会合速度定数 （kass)、 解離速度定数 （kdiss) および解離平行定数を算出した。 The measurement by the plasmon resonance method was performed using IAsys plus. A CM dextran cuvette was used as a cuvette for solid phase immobilization of ΙΤΠα. Immobilization methods and measuring methods are carried out according to pro tocol IAsys plus accompanying, 267 arc seconds The corresponding immobilized (200 arc seconds = 1 ng / mm2) 0 TOPII c of 2 · 9 to 22 · 2 nM This data was analyzed within FASTfit, and the association rate constant (kass), dissociation rate constant (kdiss) and dissociation parallel constant were calculated.
(X) アポトーシスの検出  (X) Detection of apoptosis
アポトーシスの検出は、 TUNEL法 （In situ Apoptosis Detection Kit;宝酒造 (株））、 アポトーシスによって切断された PARP を認識する抗体を用いた間接蛍 光抗体法、 DNAの ladderの検出、 Laser Scanning Cytometry (LSC; ォリンパス 光学工業 （株） ）による解析で行った。  Apoptosis can be detected by TUNEL method (In situ Apoptosis Detection Kit; Takara Shuzo Co., Ltd.), indirect fluorescent antibody method using an antibody that recognizes PARP cleaved by apoptosis, detection of DNA ladder, Laser Scanning Cytometry (LSC Olympus Optical Co., Ltd.).
(xi) アンチセンスオリゴヌクレオチドの細胞内への導入  (xi) Introduction of antisense oligonucleotide into cells
ΙΤΠα遺伝子の 1〜1 5残基に対して S- oligoアンチセンスオリゴヌクレオチ ドを設計した。 合成は、 BI0GN0STIK社に外注した。 アンチセンスの細胞内への導 入は、 Lipofectin (Gibco, BRL) を用いて行った。 1 ml の無血清培地にアンチ センスを ΙμΜになるように加えた。 他の 1 ml の無血清培地に Lipofectinを 25 μ ΐ 加えた。 両者を混合して室温で 15分間静置した。 この間、 前日に 1X106細 胞（60 培養皿）に培養した細胞を PBS (-) で 2回洗浄し、 15分後に混合溶液 を滴下した。 37°C 5% CO2インキュベーターに移して 4時間培養した。 その後、 血清入りの培地を等量加えて培養し、 24 48 72時間後に細胞の状態をチェック した。 S-oligo antisense oligonucleotides were designed for residues 1 to 15 of the ΙΤΠα gene. The synthesis was outsourced to BI0GN0STIK. Antisense was introduced into cells using Lipofectin (Gibco, BRL). Antisense was added to 1 ml of serum-free medium to a volume of {μ}. To another 1 ml of serum-free medium, 25 μl of Lipofectin was added. Both were mixed and allowed to stand at room temperature for 15 minutes. During this time, 1X10 6 The cells cultured in the cells (60 culture dishes) were washed twice with PBS (-), and the mixed solution was added dropwise 15 minutes later. The cells were transferred to a 37 ° C 5% CO 2 incubator and cultured for 4 hours. Thereafter, an equal amount of a medium containing serum was added and cultured, and the state of the cells was checked after 24 48 72 hours.
( I I ) 結果 (I I) result
( i ) ΙΤΠ αの塩基配列およびアミノ酸配列を、 図 1、 配列番号 1及び 2に示し た。  (i) The nucleotide sequence and amino acid sequence of ΙΤΠα are shown in FIG. 1, SEQ ID NOS: 1 and 2.
(ϋ) ΙΤΙΙ αの過剰発現による細胞に与える影響  (ϋ) Effect of ΙΤΙΙα overexpression on cells
ΙΤΠ ο;の Ν末端に Τ7- tagをつけて COS- 1細胞で過剰発現させ、 間接蛍光抗体 法でその局在を調べた結果、 図 2 (B)に示したように核外に局在した （ΙΤΠ αは、 FITC標識で緑色、 ΤΟΡΠ αは、 テキサスレツド標識で赤色、 核は、 Hoechst染色 で青色に表示している。）。 その際、 本来核内に存在する ΤΟΡΠ αも ΙΤΠ αと同様 に核外に存在した。 また、 ΙΤΠ αを C0S-1細胞で過剰発現させた場合、 上記に示 した細胞以外に核の断片および縮小した細胞も同時に観察された。 このような細 胞は、 アポトーシスをおこしている可能性が考えられたため、 TUNEL法およびァ ポトーシスの時に生じるポリ （ADP-リボース） ポリメラーゼ （PARP) の切断を特 異的に認識する抗体を用いて、 核の断片および縮小した細胞がアポトーシスを起 こしているかどうか検討した。 図 3 (A)に示したように TUNEL 法の結果から FITC-dUTP標識（緑色）で検出される TUNEL陽性細胞が確認された。また、図 3 (B) より、 PARPの切断も間接蛍光抗体法の結果から確認された。  As a result of overexpression in COS-1 cells with a さ せ 7-tag at the Ν-end of ΙΤΠ ο; (ΙΤΠα is green for FITC labeling, ΤΟΡΠα is red for Texas red labeling, and nuclei are blue for Hoechst staining.) At that time, ΤΟΡΠα, which was originally in the nucleus, was also outside the nucleus, as was ΙΤΠα. When ΙΤΠα was overexpressed in C0S-1 cells, nuclear fragments and reduced cells were simultaneously observed in addition to the cells shown above. These cells were considered to have apoptosis, so we used the TUNEL method and antibodies that specifically recognize the cleavage of poly (ADP-ribose) polymerase (PARP) during apoptosis. We examined whether nuclear fragments and shrunken cells were undergoing apoptosis. As shown in Fig. 3 (A), TUNEL-positive cells detected by FITC-dUTP label (green) were confirmed from the results of the TUNEL method. In addition, from FIG. 3 (B), cleavage of PARP was also confirmed from the results of the indirect fluorescent antibody method.
以上の結果から、 ΙΤΠ αを COS- 1細胞で過剰発現させた場合に、 アポトーシス を生じる細胞が検出された。 尚、 ΙΤΠ αの発現は、 抗 Τ7- tag抗体を用いたィム ノブロットで確認した。ィムノブロットの結果（図 2 (A) )より、推定分子量 38. 6k From the above results, cells that undergo apoptosis were detected when ΙΤΠα was overexpressed in COS-1 cells. The expression of ΙΤΠα was confirmed by immunoblot using an anti-Τ7-tag antibody. From the results of the immunoblot (Figure 2 (A)), the estimated molecular weight was 38.6 k
(T7-tag融合した ΙΤΠ α )より大きレ、分子量（47. 5k)で検出されたことから ΙΤΠ αの発現には、 何らかの修飾が付加されている可能性が示唆された。 The detection of で α was larger than (T7-tag fused ΙΤΠα) and detected at a molecular weight (47.5k), suggesting that some modification may be added to ΙΤΠα expression.
(i ii) 昆虫細胞を用いた ΙΤΠ aの発現系の構築と精製 ΙΤΠαの発現は、 BAC-TO-BAC Baculovirus Expression Systemsを用いて行つ た。 タンパク質の発現 （矢印） を CBB染色で確認した （図 4(A))。 精製は、 ITII α抗体カラム （実験方法参照） を用いて行った。 溶出した各フラクション （1〜8) は、透析後、 SDS- PAGE、銀染色を行ってタンパク質の精製を確認した（図 4(B))。 発現した ΙΤΠαの分子量は、 60.5kで検出され、 推定分子量 41.2k (His- tag融 合した ΙΤΠα) より大きい分子量で検出されたことから、 COS - 1細胞で発現した 時と同様に、 ΙΤΠ αは何らかの修飾が付加されている可能性が^ ¾唆された。 (i ii) Construction and purification of ΙΤΠa expression system using insect cells ΙΤΠα was expressed using BAC-TO-BAC Baculovirus Expression Systems. Protein expression (arrow) was confirmed by CBB staining (FIG. 4 (A)). Purification was performed using an ITII α antibody column (see experimental method). The eluted fractions (1 to 8) were dialyzed and subjected to SDS-PAGE and silver staining to confirm protein purification (FIG. 4 (B)). The expressed molecular weight of ΙΤΠα was detected at 60.5k, and was detected at a molecular weight larger than the estimated molecular weight of 41.2k (His-tag fused ΙΤΠα). It is suggested that some modifications may have been added.
(iv) 組み換え ΙΤΠαによる ΤΟΡΠα活性の阻害作用  (iv) Inhibition of ΤΟΡΠα activity by recombinant ΙΤΠα
昆虫細胞おょぴバキュロウィルスを用いて発現させた ΙΤΠαが、 ΤΟΡΙΙαの活 性 （Relaxation Decatenation) に対して阻害作用を示すかどうか検討した。 Decatenation活性の測定には、 基質にキネトプラスト DNA を用い （図 5(A))、 Relaxation活性の測定には、基質にスーパーコィリング DNA (図 5(B))を用いた。 活性に対する阻害効果は、 ΙΤΠαを加えなかった時の反応後の DNA量を 100%とし て ΙΤΠαを加えた時の DNA量を相対的に算出した。 ΙΤΠ αは、 ΤΟΡΠαの両活性 に対して濃度依存的に阻害した。 図 5(B)に示したように基質 DNAに ΓΠΙαを加 えても基質 DNAの電気泳動後の結果に影響を示さなかったことから、 ΙΤΠαは、 DNAと直接結合しないことが示唆された。  We examined whether ΙΤΠα expressed using insect cell baculovirus has an inhibitory effect on ΤΟΡΙΙα activity (Relaxation Decatenation). For the measurement of Decatenation activity, kinetoplast DNA was used as a substrate (FIG. 5 (A)), and for the measurement of Relaxation activity, supercoiling DNA (FIG. 5 (B)) was used as a substrate. The inhibitory effect on the activity was calculated relative to the amount of DNA when ΙΤΠα was added, with the amount of DNA after the reaction when ΙΤΠα was not added as 100%. ΙΤΠα inhibited both activities of ΤΟΡΠα in a concentration-dependent manner. As shown in FIG. 5 (B), the addition of ΓΠΙα to the substrate DNA did not affect the results after electrophoresis of the substrate DNA, suggesting that ΙΤΠα does not directly bind to DNA.
(V) 細胞周期における ΙΤΠαの発現量の推移と細胞内の局在  (V) Changes in ΙΤΠα expression during the cell cycle and intracellular localization
正常ヒ ト皮膚線維芽細胞 （NB1- RBG) を 72 時間低血清培地 （0.4% FBS、 non-essential amino acidsを含む D- MEM) で培養して、 その後 15% FBSになる ように血清を添加した。 この時点を 0時間として 4時間おきに 32時間まで細胞 を回収してレーザースキャニングサイトメーター （LSC) を用いて DNA の含有量 をヒストグラム、 および G0/G1、 S、 G2/M期の含有率を0 /0で示した （図 6(A))。 細 胞周期が正常に機能していることを確認して、 各時間での ΙΤΠαと ΤΟΡΠαの発 現を抗 ΙΤΠαおよぴ抗 ΤΟΡΠα抗体で検出し、 NIH Imageを用いてそのタンパク 質量を定量した （図 6(B))。 最大発現量を 100%として各時間の発現量を相対的に 示した。 その結果、 ΙΤΠαは Gl/S、 G2/M期を通じて GO期の約 4倍、 発現量が増 加し、 GO期を除いては常に一定レベルの発現量を有していることが明らかになつ た。 これに対して ΤΟΡΠαは、 S期後期から発現が確認され、 ΙΤΠαの発現量と ともに測定開始 32時間目で最大発現量を示した。 さらに 8時間後から 28時間ま で 4 時間ごとの ΙΤΠαと ΤΟΡΠαの細胞内の局在を間接蛍光抗体法で検出した (図 6(C))。 その結果、 ΙΤΠα は、 G1/S期には細胞質に局在し、 G2/M期から核 内に局在する細胞が認められた。 そこで、 G2/M期以降に ΙΤΠαが核内に存在す るかどう力確かめるため、 血清添カ卩 32 時間後の細胞から核分画を調製して、 核 内での ΙΤΠαの発現について抗 ΙΤΠα抗体を用いて検討した。 その結果、 調製 した核分画に ΙΤΠαの存在が確認され （図 6(D))、 細胞周期に応じて ITIIctが、 核内へ移行していることが示唆された。 この時、抗 ΙΤΠα抗体の抗原として用い たぺプチドを過剰量添加してバンドが消失したことから、 核分画で検出したバン ドが ΙΤΠαに相当することを明らかにした。 Normal human dermal fibroblasts (NB1-RBG) are cultured in low serum medium (0.4% FBS, D-MEM containing non-essential amino acids) for 72 hours, and then serum is added to 15% FBS. did. Taking this time as 0 hour, cells were collected every 4 hours up to 32 hours, and the DNA content was histogramd using a laser scanning cytometer (LSC), and the G0 / G1, S, and G2 / M phase contents were determined. 0/0 shown in (FIG. 6 (A)). After confirming that the cell cycle was functioning normally, the expression of ΙΤΠα and ΤΟΡΠα at each time was detected with anti-ΙΤΠα and anti-ΤΟΡΠα antibodies, and the protein mass was quantified using NIH Image. (Fig. 6 (B)). The expression level at each time was shown relative to the maximum expression level as 100%. As a result, expression of を 通 じ て α increased about 4 times that of the GO phase through Gl / S and G2 / M phases, In addition, it was revealed that the expression level was always constant except for the GO phase. On the other hand, expression of ΤΟΡΠα was confirmed from the late S phase, and the maximum expression level was shown 32 hours after the start of measurement together with the expression level of ΙΤΠα. Further, from 8 hours to 28 hours, intracellular localization of ΙΤΠα and ΤΟΡΠα was detected by the indirect fluorescent antibody method every 4 hours (Fig. 6 (C)). As a result, ΙΤΠα was localized in the cytoplasm during the G1 / S phase, and cells localized in the nucleus from the G2 / M phase were observed. Therefore, in order to confirm whether ΙΤΠα is present in the nucleus after the G2 / M phase, a nuclear fraction was prepared from the cells 32 hours after the addition of serum, and anti-ΙΤΠα was expressed in the nucleus. The examination was performed using antibodies. As a result, the presence of ΙΤΠα was confirmed in the prepared nuclear fraction (FIG. 6 (D)), suggesting that ITIIct translocated into the nucleus depending on the cell cycle. At this time, the band disappeared by adding an excess amount of the peptide used as the antigen of the anti-α antibody, and it was revealed that the band detected in the nuclear fraction was equivalent to Δα.
(vi) ΙΤΙΙαと ΤΟΡΠαの相互作用の検討  (vi) Examination of the interaction between ΙΤΙΙα and ΤΟΡΠα
ΙΤΠひ抗体カラムを用いて、 ΙΤΠαに ΤΟΡΠαが結合するかどうか検討した。 正常ヒ ト皮膚線維芽細胞を 72時間低血清培地で培養して、 その後 15% FBSにな るように血清を添加した。 血清添加 24、 28、 32時間後の細胞を RX buffer (実験 方法参照） 2mlに懸濁後、 超音波破砕を行い遠心分離した上***を抗体カラムに 加えた。 抗体精製と同様の方法 （実験方法参照） でカラムを洗浄して、 500 の溶出液で順次溶出した。 溶出液 （Elution l，2) は、 TCA処理後、 ΙΤΠα抗体お よび ΤΟΡΠα抗体を用いてィムノブロットを行った結果、 ΙΤΠαと ΤΟΡΠαが検 出され （図 7)、 両タンパグ質が相互作用していることが示唆された。  It was examined whether ΤΟΡΠα binds to ΙΤΠα using an antibody column. Normal human dermal fibroblasts were cultured in a low serum medium for 72 hours, and then serum was added to 15% FBS. The cells 24, 28 and 32 hours after the addition of serum were suspended in 2 ml of RX buffer (see experimental method), sonicated, centrifuged, and the supernatant was added to the antibody column. The column was washed in the same manner as in antibody purification (see Experimental method), and eluted with 500 eluates sequentially. The eluate (Elution 1, 2) was treated with TCA and subjected to immunoblotting using ΙΤΠα antibody and ΤΟΡΠα antibody. As a result, ΙΤΠα and ΤΟΡΠα were detected (Fig. 7), and both proteins interacted. It has been suggested.
TOPIIctを 2.9、 5.9、 11.6、 14.8、 17.4、 22.2nMで添加し、 ΤΟΡΠ αは濃度依 存的に、 ΙΤΠαと相互作用することを確認した （図 8 (Α))。 両者の会合速度定 数 (kass； NTS— i = 4.2 x 105)、 解離速度定数 (kdiss； x 10"3) および解 離平行定数 （KD ; M=3.8 X 10— 9) を算出した結果、 ΙΤΠαと ΤΟΡΠαの相互作用 を確認した。 TOPIIct was added at 2.9, 5.9, 11.6, 14.8, 17.4, and 22.2 nM, and it was confirmed that ΤΟΡΠα interacted with ΙΤΠα in a concentration-dependent manner (Fig. 8 (Α)). Both association rate constants (kass; NTS- i = 4.2 x 10 5), dissociation rate constants (kdiss; x 10 "3) and dissociation parallel constant (KD; M = 3.8 X 10- 9) result of calculating the The interaction between ΙΤΠα and ΤΟΡΠα was confirmed.
(vii) アンチセンスオリゴヌクレオチドの設計部位 アンチセンスオリゴヌクレオチドの配列決定は、 一般に 15〜20 mer で、 翻訳 開始の ATGコドン付近の 20 mer、 ATGから 15〜20 mer、 または第 1ェクソンと第 1 イントロンの境界を含む 20 mer が有効であることが多い。 しかし、 今回は、 genomic DNAのシークェンスがわかっていなかつたので、 ATG力 ら 15〜20 merの 塩基配列を選択した。塩基数の決定には、配列中の GC含有率が 50%以上を有し、 Gが 3つ以上連続する配列は、 アンチセンス効果以外の細胞増殖抑制効果を有し ているという報告もあることから避けた。 またアンチセンス自体がヘアピン形成 をしない配列を考慮して、 以下の 15merを選択した。 5' -TAGCAGGTCCGACAT- 3' (配 列番号 9 )。 作製したアンチセンスの細胞内への導入は、 Lipofectin を用いて行 つた。 Lipofectin によるオリゴヌクレオチドの細胞内への取り込みは、 FITC標 識のセンスオリゴヌクレオチド （アンチセンスの reverse complement) を用いて 確認した。 (vii) Design site of antisense oligonucleotide Antisense oligonucleotide sequencing is generally 15-20 mer, with 20 mer near the ATG codon for translation initiation, 15-20 mer from ATG, or 20 mer containing the boundary between the first exon and the first intron. There are many. However, this time, since the sequence of genomic DNA was not known, we selected the nucleotide sequence of 15-20 mer from ATG. In determining the number of bases, there is a report that a sequence with a GC content of 50% or more in the sequence and a sequence of three or more Gs has a cell growth inhibitory effect other than the antisense effect. Avoided from. The following 15 mer was selected in consideration of the sequence in which the antisense itself does not form a hairpin. 5'-TAGCAGGTCCGACAT-3 '(SEQ ID NO: 9). The prepared antisense was introduced into cells using Lipofectin. Incorporation of the oligonucleotide into cells by Lipofectin was confirmed using a sense oligonucleotide of FITC labeling (antisense reverse complement).
(viii)アンチセンスオリゴヌクレオチド効果による ΙΤΠ αの発現量おょぴその 局在  (viii) Expression level of ΙΤΠα by antisense oligonucleotide effect and its localization
正常ヒ ト皮膚線維芽細胞に、 Lipofectin を用いて ΙΤΠ αのアンチセンスオリ ゴヌクレオチドを導入した。 導入 48時間後、 細胞内の ΙΤΠ αのタンパク質量を ィムノブロットで検出した結果、 アンチセンス処理した細胞の ΙΤΠ αは、 コント ロールのオリゴヌクレオチドを導入した細胞のタンパク質量と比較して 66%以上 減少した（図 9 (Α) )。 また、 アンチセンスオリゴヌクレオチドの濃度依存的に ΙΤΠ αのタンパク質量は減少した（図 9 ( Β ) )。 83、 125、 167、 250ηΜのアンチ センスオリゴヌクレオチドを添加したとき、 ΙΤΙΙ αのタンパク質量は 6. 1, 47. 1、 64. 7、 83. 5%のように減少した。 さらに、 アンチセンスオリゴヌクレオチドを導 入し、 導入 12時間後に ΙΤΠ αの発現量の減少が認められ、 その際 ΤΟΡΙΙ αの発 現量は増加した（図 9 ( C ) )。 この現象は、 癌細胞 （Α549) を用いても同様の結 果が認められ、 その際、 T0PIの発現量に変化は認められなかった （図 9 (D ) )。 以上の結果より、 アンチセンスで ΙΤΠ αのタンパク質量が減少するのに伴い、 TOPII c の発現量が増加が認められた。 この時、 間接蛍光抗体法によって、 Τ0ΡΠ αの細胞内の局在を検出すると、 核から細胞質にわたって広範囲にその局在が確 認できた （図 9 ( Ε ) )。 ΙΤΠα antisense oligonucleotides were introduced into normal human skin fibroblasts using Lipofectin. 48 hours after transfection, the amount of intracellular ΙΤΠα protein was detected by immunoblot.As a result, セ ン ス α of antisense-treated cells was reduced by 66% or more compared to the amount of control oligonucleotide-transfected cells. (Fig. 9 (Α)). In addition, the amount of ΙΤΠα protein decreased in a concentration-dependent manner of the antisense oligonucleotide (FIG. 9 (Β)). When 83, 125, 167 and 250ηΜ antisense oligonucleotides were added, the protein content of ΙΤΙΙα decreased to 6.1, 47.1, 64.7 and 83.5%. Furthermore, after the antisense oligonucleotide was introduced, a decrease in the expression of ΙΤΠα was observed 12 hours after the introduction, and the expression of ΤΟΡΙΙα increased at that time (FIG. 9 (C)). A similar result was observed for this phenomenon using cancer cells () 549), and no change was observed in the expression level of T0PI (FIG. 9 (D)). From the above results, it was confirmed that the expression amount of TOPIIc increased as the amount of ΙΤΠα protein decreased with antisense. At this time, Τ0ΡΠ By detecting the intracellular localization of α, the localization was confirmed over a wide range from the nucleus to the cytoplasm (Fig. 9 (()).
(ix) 癌細胞での ΙΤΠ αと ΤΟΡΠ αの発現量の比較  (ix) Comparison of expression levels of ΙΤΠα and 発 現 α in cancer cells
ITII aの発現量を抑制することによって、 Τ0ΡΠ aの発現量が増加することが 示唆された。 ΤΟΡΠ αは、 正常細胞より癌細胞で発現量が高いことが報告されて いることから、 ΙΤΠ αの発現量は、正常細胞より癌細胞で低いことが推測された。 そこで、正常ヒ ト皮膚線維芽細胞と癌細胞 （HL60、 Jurcat , HeLa、 腎癌、 直腸癌、 胆嚢癌、大腸癌）で ΙΤΠ αと ΤΟΡΠ αのタンパク質量をィムノブ口ットで検出（図 1 0 ) し、 正常細胞の両タンパク質量を 1として、 癌細胞でのタンパク質量を相 対的に比較した （表 1 )。 腎癌での ΙΤΙΙ αのタンパク質量は、 正常細胞に比べて, 大きな差は認められなかったが、 その他、 測定した癌細胞は、 正常細胞より ΙΤΠ αのタンパク量は約 2〜3倍低く、 逆に ΤΟΡΠ αのタンパク量は 2〜3倍増加して レ、た。 表 1 ΙΠΙ aおよび ΤορΠαの発現量の相対比較  It was suggested that suppressing the expression of ITIIa would increase the expression of {0} a. Since it has been reported that ΤΟΡΠα has a higher expression level in cancer cells than in normal cells, the expression level of ΙΤΠα was presumed to be lower in cancer cells than in normal cells. Therefore, in normal human skin fibroblasts and cancer cells (HL60, Jurcat, HeLa, renal cancer, rectal cancer, gallbladder cancer, colorectal cancer), the protein levels of ΙΤΠα and ΤΟΡΠα were detected using an immunob kit (Fig. 1). However, assuming that the amount of both proteins in normal cells was 1, the amounts of proteins in cancer cells were compared relatively (Table 1). The amount of の α protein in renal cancer was not significantly different from that of normal cells.However, the amount of ΙΤΠα protein in the measured cancer cells was about 2-3 times lower than that of normal cells. Conversely, the protein amount of ΤΟΡΠα increased 2-3 times. Table 1 Comparison of expression levels of 量 a and ΤορΠα
ITII a ΤορΠα ITII a ΤορΠα
skin fibroblast (normal) 1 1  skin fibroblast (normal) 1 1
HeLa 0.32 2.7  HeLa 0.32 2.7
Jurcat 0.47 1.6  Jurcat 0.47 1.6
HL60 0.34 2.9  HL60 0.34 2.9
renal carcinoma .0.84 1.6  renal carcinoma .0.84 1.6
gallbladder carcinoma 0.58 1.8  gallbladder carcinoma 0.58 1.8
rectal carcinoma 0.60 1.9  rectal carcinoma 0.60 1.9
colon carcinoma 0.25 2.0  colon carcinoma 0.25 2.0
( x ) アンチセンスオリゴヌクレオチドによる癌細胞に対する効果 (x) Effect of antisense oligonucleotides on cancer cells
ΙΤΠ αは、 癌細胞で正常細胞より発現量は低く、 逆に ΤΟΡΠ αは、 癌細胞で正 常細胞より発現量は高かった。 さらに、 アンチセンスで正常細胞の ΙΤΠ αの発現 量を抑制すると、 T0PII aの発現量が増加することが確かめられた。 HeLa細胞と 正常ヒ ト皮膚線維芽細胞に対してアンチセンスオリゴヌクレオチドを導入し、 導 入 48 時間後の細胞を LSCで解析した結果、 正常細胞は、 GI/S arrest を起こし たが、 細胞数の減少はなかった。 これに対して、 HeLa細胞は、 細胞数が減少し、 計測した細胞の 1/3がアポトーシスを起こしていた （図 1 1 (A))。 また、 アン チセンス導入 24、 48、 69時間後の細胞を調製して DNAの ladder (図 1 1 (B))、 および 48時間後の細胞について PARPの切断有無を確かめた（図 1 1 (C))結果、 アンチセンスを導入した癌細胞は、 アポトーシスをおこし、 アンチセンスオリゴ ヌクレオチドは、癌細胞を特異的にアポトーシスに誘導する可能性が示唆された。 次に、 HeLa細胞以外の細胞で癌細胞でアンチセンスオリゴヌクレオチドによる効 果が認められるかどうかを検討した。 肺癌 （図 1 1 (D))、 大腸癌 （図 1 1 (E)) で検討したところ、 両者ともにアンチセンスオリゴヌクレオチド導入 24時間後 に明らかに細胞数の減少が認められ、 アポトーシスの指標として DNA の ladder も検出された。 今回の結果は、 癌細胞にアンチセンスを作用させると、 細胞内の ΤΟΡΠαが過剰になり、 アポトーシスを起こし、 正常細胞でアポトーシスがおこ らなかった原因として、 正常細胞では癌細胞と比較して相対的に ΤΟΡΠαの発現 量が低いため、 アンチセンス効果で ΤΟΡΠαの発現量が増加しても、 GI/Sチヱッ クポイント機構が作用して、 細胞死まで至らないのではないかと考えられる。 実施例 2 ： ΙΤΙΙαの合成べプチドによる Τ0ΡΠ aの阻害作用 ΙΤΠα was expressed at lower levels in cancer cells than in normal cells, whereas 逆 α was expressed at higher levels in cancer cells than in normal cells. Furthermore, it was confirmed that suppressing the expression of ΙΤΠα in normal cells with antisense increases the expression of T0PIIa. HeLa cells and normal human dermal fibroblasts were transfected with antisense oligonucleotides and introduced. The cells 48 hours after the injection were analyzed by LSC, and as a result, GI / Sarrest occurred in the normal cells, but the number of cells did not decrease. In contrast, the number of HeLa cells decreased, and one-third of the cells measured underwent apoptosis (Fig. 11 (A)). Cells were prepared 24, 48, and 69 hours after the introduction of antisense, and the presence or absence of PARP cleavage was confirmed for the DNA ladder (Fig. 11 (B)) and the cells 48 hours after the introduction (Fig. 11 (C )) As a result, it was suggested that cancer cells into which antisense was introduced undergo apoptosis, and that antisense oligonucleotides could specifically induce cancer cells to undergo apoptosis. Next, it was examined whether or not the effect of the antisense oligonucleotide was observed in cancer cells in cells other than HeLa cells. Examination of lung cancer (Fig. 11 (D)) and colorectal cancer (Fig. 11 (E)) revealed that both showed a clear decrease in cell number 24 hours after transfection of the antisense oligonucleotide, indicating an apoptosis index. DNA ladders were also detected. The results of this study show that when antisense is applied to cancer cells, intracellular ΤΟΡΠα becomes excessive, apoptosis occurs, and apoptosis does not occur in normal cells. Since the expression level of ΤΟΡΠα is low, the GI / S checkpoint mechanism acts to prevent cell death even if the 発 現 α expression level increases due to the antisense effect. Example 2: Inhibition of {0} a by synthetic peptide of の α
ΙΤΙΙ αの N末端側から 20残基ごとに （10残基 overlap) 合成ぺプチドを作製 し、 ΤΟΡΙΙα活性 （Relaxation Decatenation活性） の阻害作用を測定した。 な お、 ΤΟΡΠα活性は、 実施例 1の （viii) ΤΟΡΙΙαの活性測定に記載した方法と同 様にして測定した。  ぺ A synthetic peptide was prepared every 20 residues (10 residues overlap) from the N-terminal side of α, and the inhibitory effect on ΤΟΡΙΙα activity (Relaxation Decatenation activity) was measured. The ΤΟΡΠα activity was measured in the same manner as in the method described in (viii) Measurement of ΤΟΡΙΙα activity in Example 1.
その結果、 アミノ酸配列 132 〜151 (VTATFPYTTILSIWLATRRV) (配列番号 1 0) が最も強く阻害作用を示した。 結果を図 1 2に示す。 なお、 この配列以外でも TOPIIc活性を阻害する合成べプチドが認められた。 産業上の利用の可能性 本発明により、 TOPIIc と結合するタンパク質である ιτπαの生理的機能が解 明された。 本発明によれば、 ΙΤΠαの生理的機能に基づいた新規な薬剤を提供す ることが可能になり、 また、 ΤΟΡΠαと ΙΤΠαとの相互作用を利用して、 新規な 医薬品をスクリーニングする方法を提供することが可能になった。 As a result, the amino acid sequences 132 to 151 (VTATFPYTTILSIWLATRRV) (SEQ ID NO: 10) showed the strongest inhibitory action. The results are shown in FIG. In addition, other than this sequence, a synthetic peptide that inhibits TOPIIc activity was observed. Industrial applicability The present invention, the physiological function of Iotataupai alpha, a protein that binds to TOPIIc is elucidation. According to the present invention, it is possible to provide a novel drug based on the physiological function of ΙΤΠα, and to provide a method for screening a novel drug by utilizing the interaction between ΤΟΡΠα and ΙΤΠα. It became possible to do.

Claims

請求の範囲 The scope of the claims
1 . 下記の何れかのアミノ酸配列を有するヒ トトポイソメラーゼ 2 α結合因 子を含むヒ ト トポイソメラ一ゼ 2 ひ阻害剤。 1. A human topoisomerase 2 inhibitor comprising a human topoisomerase 2α binding factor having any of the following amino acid sequences:
( a ) 配列番号 1に記載のァミノ酸配列；又は  (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のアミノ酸配列において 1から数個のアミノ酸が欠失、 置換及び Z又は挿入したアミノ酸配列を有し、 ヒ ト トポイソメラーゼ 2 αと相互 作用してその活性を阻害することができるァミノ酸配列：  (b) having an amino acid sequence in which one to several amino acids are deleted, substituted, Z or inserted in the amino acid sequence of SEQ ID NO: 1, and interacts with human topoisomerase 2α to inhibit its activity. Amino acid sequences that can:
2 . 配列番号 1に記載のァミノ酸配列における部分ァミノ酸配列を有するタ ンパク質を含む、 ヒ トトポイソメラーゼ 2 α阻害剤。  2. A human topoisomerase 2α inhibitor comprising a protein having a partial amino acid sequence in the amino acid sequence of SEQ ID NO: 1.
3 . 配列番号 1 0に記載のアミノ酸配列を有するタンパク質を含む、 ヒ トト ポイソメラーゼ 2 α阻害剤。 3. SEQ ID NO: 1 0 comprising a protein having the amino acid sequence set forth in human Toto topoisomerase 2 alpha inhibitors.
4 . 下記の何れかのアミノ酸配列を有するヒ トトポイソメラーゼ 2 α結合因 子を含むアポトーシス誘導剤。 4. An apoptosis-inducing agent comprising a human topoisomerase 2α-binding factor having any one of the following amino acid sequences:
( a ) 配列番号 1に記載のァミノ酸配列；又は  (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のァミノ酸配列において 1から数個のァミノ酸が欠失、 置換及び Z又は挿入したアミノ酸配列を有し、 ヒ トトポイソメラーゼ 2 ひと相互 作用してその活性を阻害することができるァミノ酸配列：  (b) 1 to several amino acids in the amino acid sequence described in SEQ ID NO: 1 have a deleted, substituted, Z- or inserted amino acid sequence, and the human topoisomerase 2 interacts to inhibit its activity Amino acid sequences that can be:
5 . バキュロウィルスを用いて昆虫細胞で発現させた組み換えタンパク質で あることを特徴とする、 下記の何れかのアミノ酸配列を有するヒ トトポイソメラ ーゼ 2 ひ結合因子。  5. A human topoisomerase-2 binding factor having any one of the following amino acid sequences, which is a recombinant protein expressed in insect cells using a baculovirus.
( a ) 配列番号 1に記載のァミノ酸配列；又は  (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のァミノ酸配列において 1から数個のァミノ酸が欠失、 置換及び 又は挿入したアミノ酸配列を有し、 ヒ ト トポイソメラーゼ 2 αと相互 作用してその活性を阻害することができるァミノ酸配列：  (b) one or several amino acids in the amino acid sequence of SEQ ID NO: 1 have a deleted, substituted or inserted amino acid sequence, and interact with human topoisomerase 2α to inhibit its activity Amino acid sequences that can be:
6 . 下記の何れかの塩基配列中の連続する 5から 1 0 0の塩基配列のアンチ センス配列から成るアンチセンスオリゴヌクレオチド。 ( a ) 配列番号 2に記載の塩基配列列；又は 6. An antisense oligonucleotide consisting of an antisense sequence of any of the following 5 to 100 base sequences in any of the following base sequences: (a) the nucleotide sequence set forth in SEQ ID NO: 2; or
( b ) 配列番号 2に記載の塩基配列において 1から数個の塩基が欠失、 置換及び Z又は挿入した塩基配列を有し、 ヒ トトポイソメラーゼ 2 αと相互作用してその 活性を阻害することができるタンパク質をコードする塩基配列：  (b) having a nucleotide sequence in which one to several nucleotides are deleted, substituted, Z or inserted in the nucleotide sequence of SEQ ID NO: 2, and interacts with human topoisomerase 2α to inhibit its activity Nucleotide sequence encoding a protein capable of:
7 . 請求項 6に記載のアンチセンスオリゴヌクレオチドを含む、 ヒ ト トボイ ソメラーゼ 2 α結合因子の阻害剤。  7. An inhibitor of a human topoisomerase 2α-binding factor, comprising the antisense oligonucleotide according to claim 6.
8 . 請求項 6に記載のアンチセンスオリゴヌクレオチドを含む、 ヒ ト トボイ ソメラーゼ 2 αの発現増強剤。  8. An expression enhancer for human topoisomerase 2α, comprising the antisense oligonucleotide according to claim 6.
9 . 請求項 6に記載のアンチセンスオリゴヌクレオチドを含む、 アポトーシ ス誘導剤。  9. An apoptosis inducer comprising the antisense oligonucleotide according to claim 6.
1 0 . 請求項 6に記載のアンチセンスオリゴヌクレオチドを含む、 抗癌剤。 10. An anticancer agent comprising the antisense oligonucleotide according to claim 6.
1 1 . 下記の何れかのアミノ酸配列を有するヒ トトポイソメラーゼ 2 α結合 因子を認識する抗体、 又はその断片。 11. An antibody that recognizes a human topoisomerase 2α-binding factor having any of the following amino acid sequences, or a fragment thereof.
( a ) 配列番号 1に記載のァミノ酸配列；又は  (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のアミノ酸配列において 1から数個のアミノ酸が欠失、 置換及び Z又は挿入したアミノ酸配列を有し、 ヒ ト トポイソメラーゼ 2 αと相互 作用してその活性を阻害することができるァミノ酸配列：  (b) having an amino acid sequence in which one to several amino acids are deleted, substituted, Z or inserted in the amino acid sequence of SEQ ID NO: 1, and interacts with human topoisomerase 2α to inhibit its activity. Amino acid sequences that can:
1 2 . 被験物質の存在下において、 下記の何れかのアミノ酸配列を有するヒ トトポィソメラーゼ 2 "結合因子と、 ヒ トトポィソメラーゼ 2 αとの相互作用を 測定し、 該相互作用を阻害する物質を選択することを特徴とする、 ヒ トトポイソ メラーゼ 2 c結合因子の阻害剤のスクリーユング方法。 Inhibition in the presence of 1 2. Test substance, and human Toto Poi isomerase 2 "binding agent having any of the following amino acid sequences, and measuring the interaction between human Toto Poi isomerase 2 alpha, the interaction A method for screening a human topoisomerase 2c binding factor inhibitor, which comprises selecting a substance to be treated.
( a ) 配列番号 1に記載のァミノ酸配列；又は  (a) the amino acid sequence of SEQ ID NO: 1; or
( b ) 配列番号 1に記載のアミノ酸配列において 1から数個のアミノ酸が欠失、 置換及び Z又は挿入したアミノ酸配列を有し、 ヒ ト トポイソメラ一ゼ 2 αと相互 作用してその活性を阻害することができるァミノ酸配列： (B) several amino acids are deleted from 1 in the amino acid sequence set forth in SEQ ID NO: 1, have a substituent and Z or inserted amino acid sequence, inhibit its activity and interact with the human Topoisomera Ichize 2 alpha Amino acid sequences that can be:
1 3 . 被験物質が、 低分子化合物、 抗体、 オリゴヌクレオチド、 又はそれら のライブラリーである、 請求項 1 2に記載の方法。 13. The method according to claim 12, wherein the test substance is a low-molecular compound, an antibody, an oligonucleotide, or a library thereof.
14. 請求項 1 2又は 1 3に記載の方法により得られる、 ヒ トトポイソメラ ーゼ 2ひ結合因子の阻害剤。 14. An inhibitor of human topoisomerase-2 binding factor, obtained by the method according to claim 12 or 13.
PCT/JP2002/006415 2001-06-27 2002-06-26 Novel human topoisomerase 2α inhibitory protein and utilization thereof WO2003002737A1 (en)

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